TWI747866B - Inactivation of pathogens by delipidation - Google Patents

Inactivation of pathogens by delipidation Download PDF

Info

Publication number
TWI747866B
TWI747866B TW105144117A TW105144117A TWI747866B TW I747866 B TWI747866 B TW I747866B TW 105144117 A TW105144117 A TW 105144117A TW 105144117 A TW105144117 A TW 105144117A TW I747866 B TWI747866 B TW I747866B
Authority
TW
Taiwan
Prior art keywords
mbcd
mixture
virus
hours
viruses
Prior art date
Application number
TW105144117A
Other languages
Chinese (zh)
Other versions
TW201733615A (en
Inventor
萊拉 戴思
史帝芬 吳
Original Assignee
美商益農美國公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 美商益農美國公司 filed Critical 美商益農美國公司
Publication of TW201733615A publication Critical patent/TW201733615A/en
Application granted granted Critical
Publication of TWI747866B publication Critical patent/TWI747866B/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/10011—Arenaviridae
    • C12N2760/10051—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16151—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/10011—Arteriviridae
    • C12N2770/10051—Methods of production or purification of viral material

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Pulmonology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The present invention relates to vaccines containing enveloped viruses whose envelopes have been depleted of lipids using methyl β-cyclodextrin (MBCD). Delipidation of enveloped viruses in a two-step process abolishes the infectivity of those viruses, which allows delipidated viruses to be used safely in vaccines. Use of MBCD to deplete lipids such as cholesterol, in contrast to other methods, results in viruses with less than 20% of the cholesterol of an untreated virus but such delipidated viruses retain at least 85% of the protein content of an untreated virus. Delipidation by MBCD also preserves the immunogenicity of the viral proteins. The present invention of using MBCD treatment in a two-step process to delipidate enveloped virus represents a new alternative for generation of inactivated viruses which are incorporated into effective vaccines.

Description

藉由去脂質化使病原體不活化Inactivate pathogens by delipidation

本發明係關於含有包膜病毒粒子之疫苗，該等包膜病毒粒子之包膜已使用甲基β-環糊精(MBCD)去脂質化。The present invention relates to vaccines containing enveloped virus particles. The envelope of the enveloped virus particles has been delipidated using methyl β-cyclodextrin (MBCD).

病毒為其自身不具有代謝活性之可移動遺傳元件。實際上，病毒必須感染宿主細胞且使用該宿主細胞之能量及程序來繁殖。一些病毒為「裸」病毒，意謂個別病毒粒子(或病毒體)僅包含包圍病毒基因組之衣殼及可能的少許病毒編碼非結構蛋白質。在其他病毒中，衣殼含於膜或包膜內。由於病毒基因組通常太小而無法編碼參與脂質合成之蛋白質，故此類包膜病毒必須由宿主細胞脂質建構其包膜。由此，病毒包膜之脂質含量視宿主細胞之脂質特性而變化。 病毒良好地適應其宿主生物體且常常為致病的，導致其宿主患病且甚至死亡。病毒可藉由感染人類及/或人類之伴侶動物抑或藉由影響作為食物來源之植物及動物而具有巨大的經濟影響。疫苗為保護宿主動物免受病毒感染之較佳方法。疫苗產生對病毒之保護性免疫反應，包含抗體及細胞免疫。通常需要兩種類型之反應來減少對寄主細胞之新感染且自宿主清除病毒粒子及受感染細胞。 約40年前，Moore等人(J. Virol. 27(2): 320 - 329, 1978)證實：使用膽固醇自病毒包膜去除去脂質之不含膽固醇的囊泡可降低病毒粒子感染其目標細胞之能力。對比而言，其他研究者報導病毒感染性需要膽固醇存在於靶細胞膜中但不存在於病毒包膜中。還有其他研究者假定，感染性減弱之原因為移除膽固醇之過程，而非包膜中缺乏膽固醇。不管膽固醇在病毒功能中之作用如何，尚未證實去膽固醇病毒完全且永久性地喪失感染性，亦尚未顯示去膽固醇病毒適用於安全且有效的疫苗。 已報導去除膽固醇之化學手段，諸如使用環糊精、正丁醇、二異丙醚(DIPE)、氟醚(諸如，七氟烷)、界面活性劑(諸如，TRITON X-100™ (聚乙二醇對(1,1,3,3-四甲基丁基)-苯醚或聚氧乙烯辛基苯醚)或TWEEN 20™ (PEG (20)脫水山梨糖醇單月桂酸酯))、乙醚及其組合。已證明一些化學手段過於苛刻，導致病毒體損失病毒蛋白質或剩餘蛋白質結構變化。蛋白質之存在及天然構型將係必需的，以便使去膽固醇病毒體適用於疫苗，特定言之刺激產生針對於病毒包膜蛋白質之中和抗體的疫苗中。 在一些狀況下，已顯示膽固醇之去除為可逆的。添加外源性膽固醇可復原病毒包膜且恢復感染性。此可造成關於在疫苗中使用去膽固醇病毒之安全問題，此係因為血液或其他體液中或甚至與去膽固醇病毒體接觸之宿主細胞膜中存在的膽固醇及其他脂質可潛在地復原病毒體且恢復感染性。 通常認為含有減毒病毒之疫苗在產生保護性免疫方面係最有效的。然而，存在一個安全問題，即減毒病毒可突變或重組且由此回復至病毒性狀態。通常認為在疫苗中使用不活化(亦即，經殺滅)病毒更安全，但由於去活化手段常常更改或破壞抗原性抗原決定基，因此降低或失去充當保護性免疫之刺激劑的效力。 由於缺乏有效疫苗，故包膜病毒持續對人類及非人類動物造成健康風險，且包膜病毒引起之疾病具有巨大的經濟影響。需要既安全且有效之經改良疫苗。舉例而言，保留病毒抗原之免疫原性、維持病毒體完好且造成極少或無安全風險之使包膜病毒不活化的經改良方法將有益於緩解由包膜病毒引起之疾病。本申請案揭示病毒去活化之此類經改良方法，包含用MBCD對病毒包膜進行去脂質化。Viruses are mobile genetic elements that have no metabolic activity by themselves. In fact, the virus must infect a host cell and use the energy and processes of the host cell to reproduce. Some viruses are "naked" viruses, meaning that individual virus particles (or virions) only contain the capsid surrounding the viral genome and possibly a few virus-encoded non-structural proteins. In other viruses, the capsid is contained in the membrane or envelope. Since viral genomes are usually too small to encode proteins involved in lipid synthesis, such enveloped viruses must construct their envelopes from host cell lipids. Therefore, the lipid content of the virus envelope varies depending on the lipid characteristics of the host cell. Viruses adapt well to their host organisms and are often pathogenic, causing their hosts to become sick and even die. Viruses can have a huge economic impact by infecting humans and/or human companion animals or by affecting plants and animals that are food sources. Vaccines are a better way to protect host animals from viral infections. The vaccine produces a protective immune response to the virus, including antibodies and cellular immunity. Two types of responses are generally required to reduce new infections of host cells and to clear virus particles and infected cells from the host. About 40 years ago, Moore et al. (J. Virol. 27(2): 320-329, 1978) proved that the use of cholesterol to remove lipid-free cholesterol-free vesicles from the viral envelope can reduce viral particles from infecting their target cells The ability. In contrast, other researchers have reported that viral infectivity requires cholesterol to be present in the target cell membrane but not in the viral envelope. Other researchers have hypothesized that the reason for the reduced infectivity is the process of removing cholesterol, not the lack of cholesterol in the envelope. Regardless of the role of cholesterol in viral functions, it has not been proven that cholesterol-removing viruses completely and permanently lose infectivity, and it has not been shown that cholesterol-removing viruses are suitable for safe and effective vaccines. Chemical methods for removing cholesterol have been reported, such as the use of cyclodextrin, n-butanol, diisopropyl ether (DIPE), fluoroether (such as sevoflurane), surfactants (such as, TRITON X-100™ (polyethylene Glycol (1,1,3,3-tetramethylbutyl)-phenyl ether or polyoxyethylene octyl phenyl ether) or TWEEN 20™ (PEG (20) sorbitan monolaurate)), Ether and combinations thereof. It has been proved that some chemical methods are too harsh, causing the virion to lose the viral protein or change the structure of the remaining protein. The presence and natural configuration of the protein will be necessary in order to make the cholesterol-removing virosomes suitable for vaccines, specifically to stimulate the production of vaccines directed against neutralizing antibodies to the viral envelope proteins. In some cases, the removal of cholesterol has been shown to be reversible. Adding exogenous cholesterol can restore the virus envelope and restore infectivity. This can cause safety issues regarding the use of cholesterol-removing viruses in vaccines, because cholesterol and other lipids present in blood or other body fluids or even in the host cell membranes that are in contact with cholesterol-removing virions can potentially restore virions and restore infection sex. It is generally believed that vaccines containing attenuated viruses are the most effective in generating protective immunity. However, there is a safety issue that the attenuated virus can mutate or recombine and thereby revert to a viral state. It is generally believed that it is safer to use inactivated (that is, killed) viruses in vaccines, but because deactivation means often alter or destroy antigenic epitopes, the effectiveness of acting as a stimulant for protective immunity is reduced or lost. Due to the lack of effective vaccines, enveloped viruses continue to pose health risks to humans and non-human animals, and diseases caused by enveloped viruses have a huge economic impact. There is a need for improved vaccines that are both safe and effective. For example, an improved method that preserves the immunogenicity of viral antigens, maintains the virions intact, and causes little or no safety risk to inactivate enveloped viruses will be beneficial to alleviate diseases caused by enveloped viruses. This application discloses such an improved method of virus deactivation, including delipidizing the virus envelope with MBCD.

因此，本發明提供一種用於製備不活化包膜病毒之方法，該方法包含：將包含包膜病毒之溶液與第一MBCD溶液混合，獲得第一混合物；培育該第一混合物第一時間段；將來自該第一混合物之該包膜病毒與第二MBCD溶液混合，獲得第二混合物；及培育該第二混合物第二時間段。該第一混合物中MBCD的濃度為至少5 mM至約100 mM。進一步而言，該第一混合物中MBCD的濃度為約20 mM至約40 mM。更進一步而言，該第一混合物中MBCD的濃度為約20 mM、約30 mM或約40 mM。 本發明提供一種用於製備不活化包膜病毒之方法，該方法包含：將包含包膜病毒之溶液與第一MBCD溶液混合，獲得第一混合物；培育該第一混合物第一時間段；將來自該第一混合物之該包膜病毒與第二MBCD溶液混合，獲得第二混合物；及培育該第二混合物第二時間段；其中該第二混合物中MBCD的濃度為至少10 mM至約100 mM。進一步而言，該第二混合物中MBCD的濃度為約30 mM至約50 mM。更進一步而言，該第二混合物中MBCD的濃度為約30 mM、約40 mM或約50 mM。 本發明提供一種用於製備不活化包膜病毒之方法，該方法包含：將包含包膜病毒之溶液與第一MBCD溶液混合，獲得第一混合物；培育該第一混合物第一時間段；將來自該第一混合物之該包膜病毒與第二MBCD溶液混合，獲得第二混合物；及培育該第二混合物第二時間段；其中該第一時間段為約15分鐘至約24小時。進一步而言，該第一時間段為約4小時至約24小時。更進一步而言，該第一時間段為約4、6、8、10、12、14、16、18、20、22或24小時。 本發明提供一種用於製備不活化包膜病毒之方法，該方法包含：將包含包膜病毒之溶液與第一MBCD溶液混合，獲得第一混合物；培育該第一混合物第一時間段；將來自該第一混合物之該包膜病毒與第二MBCD溶液混合，獲得第二混合物；及培育該第二混合物第二時間段；其中該第二時間段為約4小時至約48小時。進一步而言，該第二時間段為約24小時至約48小時。更進一步而言，該第二時間段為約24、30、36、40、44或48小時。 本發明提供一種用於製備不活化包膜病毒之方法，該方法包含：將包含包膜病毒之溶液與第一MBCD溶液混合，獲得第一混合物；培育該第一混合物第一時間段；將來自該第一混合物之該包膜病毒與第二MBCD溶液混合，獲得第二混合物；及培育該第二混合物第二時間段；其中在第一時間段期間該第一混合物之溫度為室溫或約20℃至約25℃。進一步而言，在第一時間段期間該第一混合物之溫度為約22℃至約24℃。 本發明提供一種用於製備不活化包膜病毒之方法，該方法包含：將包含包膜病毒之溶液與第一MBCD溶液混合，獲得第一混合物；培育該第一混合物第一時間段；將來自該第一混合物之該包膜病毒與第二MBCD溶液混合，獲得第二混合物；及培育該第二混合物第二時間段；其中在第二時間段期間該第二混合物之溫度為室溫或約20℃至約25℃。進一步而言，在第二時間段期間該第二混合物之溫度為約22℃至約24℃。 本發明提供一種用於製備不活化包膜病毒之方法，該方法包含：將包含包膜病毒之溶液與第一MBCD溶液混合，獲得第一混合物；培育該第一混合物第一時間段；將來自該第一混合物之該包膜病毒與第二MBCD溶液混合，獲得第二混合物；及培育該第二混合物第二時間段；其中在獲得該第二混合物之步驟之前將該包膜病毒與該第一混合物分離。或者且非限制性地，第二MBCD溶液可與第一混合物直接混合。可在與第一混合相同的不活化容器中進行該直接混合，或第一混合物先移至一個新的不活化容器後再與第二MBCD溶液混合。 本發明提供一種用於製備不活化包膜病毒之方法，該方法包含：將包含包膜病毒之溶液與第一MBCD溶液混合，獲得第一混合物；培育該第一混合物第一時間段；將來自該第一混合物之該包膜病毒與第二MBCD溶液混合，獲得第二混合物；及培育該第二混合物第二時間段；其中該第一時間段進一步包含混合該第一混合物。攪拌可為約30 rpm至約100 rpm。較佳地，攪拌可為約40 rpm至約60 rpm。最佳地，攪拌可為約50 rpm。 本發明提供一種用於製備不活化包膜病毒之方法，該方法包含：將包含包膜病毒之溶液與第一MBCD溶液混合，獲得第一混合物；培育該第一混合物第一時間段；將來自該第一混合物之該包膜病毒與第二MBCD溶液混合，獲得第二混合物；及培育該第二混合物第二時間段；其中該第二時間段進一步包含混合該第二混合物。攪拌可為約30 rpm至約100 rpm。較佳地，攪拌可為約50 rpm。 本發明提供一種用於製備不活化包膜病毒之方法，該方法包含：將包含包膜病毒之溶液與第一MBCD溶液混合，獲得第一混合物；培育該第一混合物第一時間段；將來自該第一混合物之該包膜病毒與第二MBCD溶液混合，獲得第二混合物；及培育該第二混合物第二時間段；其中該包膜病毒為豬繁殖與呼吸症候群(PRRS)病毒。該包膜病毒亦可為豬流行性下痢病毒(PEDV)。 本發明提供一種去脂質包膜病毒，其藉由以下方法獲得：將包含包膜病毒之溶液與第一MBCD溶液混合，獲得第一混合物；培育該第一混合物第一時間段；將來自該第一混合物之該包膜病毒與第二MBCD溶液混合，獲得第二混合物；及培育該第二混合物第二時間段。較佳地，該第一混合物中MBCD的濃度為至少5 mM至約100 mM，或約20 mM至約40 mM。較佳地，該第二混合物中MBCD的濃度為至少10 mM至約100 mM，或約30 mM至約50 mM，或約30 mM、約40 mM或約50 mM。較佳地，該第一時間段為約15分鐘至約24小時，或約4小時至約24小時，或約4、6、8、10、12、14、16、18、20、22或24小時。較佳地，該第二時間段為約4小時至約48小時，或約24小時至約48小時，或約24、30、36、40、44或48小時。較佳地，在第一時間段期間該第一混合物之溫度為室溫，或約20℃至約25℃，或甚至約22℃至約24℃。較佳地，在第二時間段期間該第二混合物之溫度為室溫，或約20℃至約25℃，或甚至約22℃至約24℃。較佳地，在獲得第二混合物之步驟之前將包膜病毒與第一混合物分離。或者且非限制性地，第二MBCD溶液可與第一混合物直接混合。可在與第一混合相同的不活化容器中進行該直接混合，或第一混合物先移至一個新的不活化容器後再與第二MBCD溶液混合。較佳地，該第一時間段進一步包含混合該第一混合物，其中該攪拌可為約30 rpm至約100 rpm，約40 rpm至約60 rpm，或約50 rpm。較佳地，該第二時間段進一步包含混合該第二混合物，其中攪拌可為約30 rpm 至約100 rpm，約40 rpm至約60 rpm，或約50 rpm。較佳地，該包膜病毒為豬繁殖與呼吸症候群(PRRS)病毒或豬流行性下痢病毒(PEDV)。 本發明提供一種包含去脂質包膜病毒的疫苗，該去脂質包膜病毒藉由以下方法獲得：將包含包膜病毒之溶液與第一MBCD溶液混合，獲得第一混合物；培育該第一混合物第一時間段；將來自該第一混合物之該包膜病毒與第二MBCD溶液混合，獲得第二混合物；及培育該第二混合物第二時間段。較佳地，該第一混合物中MBCD的濃度為至少5 mM至約100 mM，或約20 mM至約40 mM。較佳地，該第二混合物中MBCD的濃度為至少10 mM至約100 mM，或約30 mM至約50 mM，或約30 mM、約40 mM或約50 mM。較佳地，該第一時間段為約15分鐘至約24小時，或約4小時至約24小時，或約4、6、8、10、12、14、16、18、20、22或24小時。較佳地，該第二時間段為約4小時至約48小時，或約24小時至約48小時，或約24、30、36、40、44或48小時。較佳地，在第一時間段期間該第一混合物之溫度為室溫，或約20℃至約25℃，或甚至約22℃至約24℃。較佳地，在第二時間段期間該第二混合物之溫度為室溫，或約20℃至約25℃，或甚至約22℃至約24℃。較佳地，在獲得第二混合物之步驟之前將包膜病毒與第一混合物分離。或者且非限制性地，第二MBCD溶液可與第一混合物直接混合。可在與第一混合相同的不活化容器中進行該直接混合，或第一混合物先移至一個新的不活化容器後再與第二MBCD溶液混合。較佳地，該第一時間段進一步包含混合該第一混合物，其中攪拌可為約30 rpm至約100 rpm，約40 rpm至約60 rpm，或約50 rpm。較佳地，該第二時間段進一步包含混合該第二混合物，其中攪拌可為約30 rpm至約100 rpm，或約50 rpm。較佳地，該包膜病毒為豬繁殖與呼吸症候群(PRRS)病毒或豬流行性下痢病毒(PEDV)。 本發明提供一種包含去脂質包膜病毒之疫苗，該去脂質包膜病毒藉由以下方獲得：將包含包膜病毒之溶液與第一MBCD溶液混合，獲得第一混合物；培育該第一混合物第一時間段；將來自該第一混合物之該包膜病毒與第二MBCD溶液混合，獲得第二混合物；及培育該第二混合物第二時間段；其中該疫苗進一步包含佐劑、穩定劑、防腐劑及摻合稀釋劑中之至少一者。較佳地，該佐劑為水性聚合物佐劑，其中該聚合物為丙烯酸酯或聚丙烯酸酯。較佳地，該穩定劑包含糖、碳水化合物、蛋白質及明膠中之至少一者。較佳地，該防腐劑為延遲、抑制或妨礙微生物之生長、代謝活動或增殖的抗生素或生物穩定化合物。較佳地，該摻合稀釋劑為水、磷酸鹽緩衝鹽水、細胞培養基或包含生理鹽度及pH之其他溶液。 本發明提供一種去脂質包膜病毒之用途，該去脂質包膜病毒藉由以下方法獲得：將包含包膜病毒之溶液與第一MBCD溶液混合，獲得第一混合物；培育該第一混合物第一時間段；將來自該第一混合物之該包膜病毒與第二MBCD溶液混合，獲得第二混合物；及培育該第二混合物第二時間段；其中該用途包含治療由包膜病毒引起之疾病或症狀。 本發明提供一種去脂質包膜病毒之用途，其係用於製造用於治療由包膜病毒引起之疾病或症狀的藥劑；其中該去脂質包膜病毒藉由以下方法獲得：將包含包膜病毒之溶液與第一MBCD溶液混合，獲得第一混合物；培育該第一混合物第一時間段；將來自該第一混合物之該包膜病毒與第二MBCD溶液混合，獲得第二混合物；及培育該第二混合物第二時間段。 如本文所使用，「包膜病毒」為具有包圍核蛋白核心之脂質雙層膜或包膜的任何病毒。病毒包膜通常衍生自宿主細胞膜且含有磷脂、糖脂、鞘脂及固醇(諸如，膽固醇)。包膜病毒包括具有由DNA或RNA編碼之基因組的病毒。包膜病毒之種類包括(但不限於)：疱疹病毒、痘病毒、虹彩病毒(iridovirus)、肝炎病毒、反轉錄病毒、正黏液病毒、沙粒狀病毒、布尼亞病毒(bunyaviruses)、副黏液病毒、單股反鏈病毒、棒狀病毒、絲狀病毒、冠狀病毒、動脈炎病毒、黃病毒屬及披膜病毒。正不斷地發現新的包膜病毒，且包膜病毒被分類並且隨著每一病毒得到更佳表徵而再分類。可在諸如Fields Virology (D.M. Knipe及P.M. Howley編，Lippincott Williams & Wilkins, Philadelphia, PA, USA, 2013) (現發行其第六版)之正文中找到包膜病毒之更完整描述。 如本文所使用之術語「病毒」可意謂病毒種類或可互換地意謂個別感染性單元或含有核酸、蛋白質及包膜之單元。「病毒粒子」為個別感染性單元，且此術語與術語「病毒體」同義。 「去脂質化」為自病毒包膜移除或去除脂質之過程。藉由用MBCD自病毒粒子移除之膽固醇的量來量測去脂質化。然而，MBCD亦可提取、移除或去除其他脂質，諸如磷脂。儘管本文中所述之過程由於MBCD介導之去脂質化而將病毒蛋白質之蛋白損失減至最少，但亦顯示MBCD與一些蛋白質相互作用。 「感染性」為病毒在宿主細胞內產生感染之能力，該宿主細胞為來自人類或非人類動物之能夠支援病毒複製的任何細胞。可(例如)藉由受一定數目的病毒粒子感染之宿主細胞的數目或百分比，或藉由感染宿主細胞所需之病毒粒子的數目或百分比來量測感染性。可以可操作方式計數病毒粒子，諸如藉由測定一定體積之病毒粒子內的溶菌斑形成單元(PFU)的數目。亦可以物理方式量測病毒粒子，諸如藉由所偵測之特定病毒蛋白質之存在，藉由酶聯結免疫吸附分析法(ELISA)，或藉由量測溶液中所存在之病毒基因組的複本數目(例如藉由即時定量聚合酶鏈式反應(qPCR)偵測)。 「抗原」為能夠被生物體之免疫系統特異性地偵測到之任何分子。典型地，病毒抗原為由病毒基因組編碼之病毒蛋白質。病毒抗原之存在可被T淋巴細胞及B淋巴細胞兩者特異性地偵測到。「免疫原性」係指抗原誘發免疫反應之能力。對疫苗而言，病毒抗原之免疫原性將較佳地在動物中引起將減少、緩和或減輕病毒感染之保護性免疫。 與抗原相比而言，「佐劑」為免疫反應之非特異性刺激劑。佐劑可藉由結合模式識別受體(PRR)且使其活化來刺激先天性免疫反應。舉例而言，該等PRR刺激劑可為病毒或細菌核酸、來自細菌或寄生蟲之脂質，或細菌蛋白質或毒素，或該等分子之任何人工構建模擬物。佐劑亦包括(但不限於)：聚集抗原以促進B淋巴細胞之識別或吞噬細胞之吸收的無機化合物，諸如礬、氫氧化鋁、磷酸鋁、磷酸鈣氫氧化物或硫酸銨；油；以及清潔劑。佐劑亦可為免疫信號傳導之宿主介體，諸如(但不限於)細胞激素、淋巴激素、趨化激素、干擾素、過敏毒素、生長因子、分化因子及黏附分子。 如本文中所使用，術語「治療(treating/to treat/treatment)」包括抑制、延緩、終止、減輕、減緩或逆轉現有症狀、病症、病狀或疾病之進展或嚴重程度。可防治性地或治療性地施加治療。 如本文所用，「向動物投予」包括經皮膚、經皮下、肌肉內、經黏膜、經黏膜下層、經皮、經口或鼻內投與。投與可包括注射或表面投與。 以下實驗實例說明去脂質化製程。應瞭解，其他實施例及用途對熟習此項技術者而言將為顯而易見的，且本發明不限於此等特定說明性實例或較佳實施例。例如(但不限於)視包膜病毒及包膜病毒在其中進行複製之宿主細胞的類型及種類而定，包膜病毒在其包膜中含有不同水準之膽固醇。如熟習此項技術者將瞭解，本文中所述之去脂質化製程可能需要對每一特定病毒原液進行最佳化。微生物之膜可含有膽固醇、類藿烷或其他固醇及類固醇分子，且因此不為病毒之病原體亦可藉由本文中所揭示之方法不活化。實例 1 使用以下程序對人類流感病毒H1N1 A/WSN/33病毒株進行去脂質。在以下實例中，流感病毒(IFV)之去脂質化削弱感染性。由於本發明之去脂質化方法保留病毒包膜蛋白質，故預期接種去脂質IFV之動物產生保護其免受致病劑量之毒性病毒感染的免疫原性反應。試劑 ： 人類流感病毒H1N1 A/WSN/33病毒株及其宿主細胞株MDCK可購自Wuxi AppTec (中國，上海)。其他試劑包括MEM (Invitrogen, Carlsbad, CA)；EMEM (Sigma-Aldrich, St. Louis, MO)；ULTRAMDCK™無血清培養基(Lonza, Inc., Allendale, NJ)；胎牛血清(FBS；Invitrogen)；0.25%胰蛋白酶-EDTA (乙二胺四乙酸；Invitrogen)；甲基-β-環糊精(MBCD，Sigma-Aldrich)；AMPLEX Red膽固醇分析套組(Invitrogen)；PIERCE™ BCA蛋白質分析套組(Rockford，IL)；及MTT (Sigma-Aldrich)。H1N1 A / WSN / 33 流感病毒在 MDCK 細胞株中之 增殖： 根據以下程序用IFV接種細胞。自在T-75燒瓶中生長之MDCK細胞中移除培養基，在室溫下用5 mL PBS沖洗MDCK細胞單層，且在37℃下用1.5 mL胰蛋白酶/EDTA分離細胞。將細胞再懸浮於含有10% FBS之10 mL MEM中，且藉由在4℃下以800 rpm離心5分鐘而集結成塊。將細胞再懸浮於含有10% FBS之MEM中，並且將細胞密度調整至2.5 × 105 個細胞/毫升。將十五毫升之MDCK細胞懸浮液接種至各T-75燒瓶中，且將燒瓶在37℃以及5% CO2 下放置並且培育隔夜。 當MDCK細胞匯合超過90%時，自T75燒瓶移除MEM，添加含有1% FBS及1 μg/mL胰蛋白酶之5 mL EMEM維持液，且以0.01之感染倍率(MOI)用流感病毒感染細胞。在37℃以及5% CO2 下培育燒瓶60分鐘至90分鐘，且每隔15分鐘輕輕地振盪。向各燒瓶中添加含有1% FBS及1 μg/mL胰蛋白酶之十毫升EMEM維持液，並且在37℃及5% CO2 下培育燒瓶48小時。在達成病毒之大致80%細胞病變效應(CPE)時(通常在約48小時後)，收集培養物上清液以獲得IFV。H1N1 A / WSN / 33 IFV 之 純化及滴定 ： 根據以下程序純化IFV。以3,000 rpm離心所採集之培養物上清液20分鐘以移除細胞碎屑，且採集上清液。以38,000 rpm離心該上清液60分鐘，以使IFV集結成塊。將IFV離心塊再懸浮於適當體積之生理緩衝鹽水(PBS)中，得到＞1.0 × 109 溶菌斑形成單元/mL (PFU/mL)之最終力價。在-80℃下儲存100 μL此經濃縮IFV儲備溶液之等分試樣。 使用以下程序針對活體外感染性滴定IFV。使用上述細胞培養物及轉移程序，以2.5 × 105 個細胞/毫升之最終密度將MDCK細胞懸浮於含有10% FBS之MEM中。向6孔培養盤之各孔中添加二毫升之MDCK細胞懸浮液，並且在37℃以及5% CO2 下培育培養盤隔夜。在37℃水浴中使病毒儲備液解凍，之後在4℃下以500 ×g 離心10分鐘。使用ULTRAMDCK™無血清培養基及2.5 μg/mL胰蛋白酶作為稀釋緩衝液，製備儲備溶液之1/10稀釋系列並且在使用之前在4℃下進行儲存。 當細胞匯合至少90%時，移除培養基，且向各孔中添加0.5 mL稀釋緩衝液及0.5 mL病毒稀釋液。對於陰性對照組，使用1.0 mL稀釋緩衝液。在添加各稀釋液之後立刻輕輕振盪培養盤。在37℃下、在5% CO2 中培育培養盤60分鐘至90分鐘，且每隔15分鐘搖動培養盤。接著，在37℃下用含2.5%低熔點瓊脂糖之PBS溶液與稀釋緩衝液之3 mL的1:4混合物覆蓋各6孔培養盤之各孔。在室溫下培育培養盤15分鐘以使覆層混合物固化；接著在37℃以及5% CO2 下培育培養盤3天。 使用以下程序使溶菌斑顯色。在溶菌斑充分形成之後(感染後3天)，添加1 mL 4%多聚甲醛，且在室溫下培育培養盤1小時。棄去溶解的瓊脂糖，且向各孔中添加0.1 mL之0.5%結晶紫。在培育15分鐘之後對溶菌斑數目進行計數，且基於稀釋因數轉換為力價。去脂質 化 ： 在兩個並行反應中進行經純化IFV之去脂質化程序：溶劑處理，其包含使IFV與MBCD接觸；以及模擬處理，其中IFV以相同方式加以處理但不暴露於MBCD。在模擬處理及溶劑處理兩者中使用相同力價的IFV。可滴定模擬處理中之IFV，以得到在溶劑處理之後剩餘的去脂質IFV之量的間接量測值。 根據以下程序進行溶劑處理及模擬處理。在艾本德(Eppendorf)試管中將上文所製備之IFV儲備液之等分試樣(100 μL) (＞1.0 × 109 PFU/mL)稀釋至900 μL PBS中，以達成1.0 - 5.0 × 108 PFU/mL之最終力價。對於溶劑處理，添加含MBCD之PBS，達至50 mM之最終濃度，或向模擬處理中添加相同體積之PBS。隨後用封口膜封蓋且密封艾本德試管。將所有樣本固持在預加熱至37℃之SHZ-82恆溫定軌氣浴震盪器(中國，Changzhou Guohua Appliance Co.)中。在37℃下使經溶劑處理樣本及經模擬處理樣本以220 rpm之定軌旋轉速度旋轉30分鐘或45分鐘。在微離心機中使樣本旋轉1分鐘，且將含有IFV之上清液轉移至超離心機試管。以200,000 ×g (OPTIMATM L-100XP；Beckman Coulter, Inc.，Indianapolis IN)離心試管30分鐘，以使IFV集結成塊，且棄去上清液。將IFV離心塊再懸浮於上文添加至每一艾本德試管之1/10初始體積中，並且在進一步分析之前在-80℃下進行儲存。去脂質 IFV 之表徵： 根據製造商的說明使用BCA分析(PIERCE™ BCA蛋白質分析套組)來測定去脂質及經模擬處理之IFV的蛋白質含量。使用根據製造商之說明進行的膽固醇分析(AMPLEX® Red膽固醇分析套組，Life Technologies，Grand Island，NY)來測定膽固醇含量。使用與如上文所闡述相同的滴定程序來量測活體外感染性。 如下測定血球凝集(HA)活性。用PBS洗滌新鮮分離的雞血三次，且使紅細胞(RBC) 再懸浮於濃度為1%之PBS中。在PBS中製得病毒稀釋液(50 μL)，且將該等稀釋液與50 μL之RBC懸浮液混合。向96孔培養盤中之個別孔中添加混合物，且使RBC靜置45分鐘。若存在RBC斑點或集結粒，則判定孔呈HA陰性(亦即，無RBC凝集)，且若存在平滑的RBC懸浮液，則判定孔呈陽性。 根據上文所述之程序製備兩個批次的去脂質及經模擬處理之IFV樣本。用於測試之IFV儲備液具有2.2 × 1010 PFU/mL之力價。在第一批次(「製劑A」)中，去脂質化時間為30分鐘；在第二批次(「製劑B」)中，去脂質化時間為45分鐘。在超離心之後，表徵去脂質及經模擬處理之樣本。 活體外感染性展示：去脂質樣本之PFU為對應模擬樣本之PFU的約1/7,000。感染性結果示於下表1中。儘管存在力價差異，但各種樣本之蛋白質含量在彼此的10%內。來自製劑A之去脂質IFV的蛋白質含量為模擬樣本的97%；且來自製劑B之去脂質IFV的蛋白質含量為模擬樣本的93% (參見 表1)。亦觀測到，在處理後，大致20%之起始物質恢復，如藉由蛋白質含量所測定。基於蛋白質含量之恢復百分比可與起始物質(2.2 × 109 PFU/mL)及經模擬處理樣本(4.0 × 108 PFU/mL，其為起始力價的18%)之經量測力價相比。與模擬處理相比，在去脂質化之後，少於2%之膽固醇保留(參見 表1)。 表1

Figure 105144117-A0304-0001
註：a 與模擬處理相比之感染性分數；b 與模擬處理相比之百分比。 模擬處理表明，物理處理(震盪及離心)造成力價及蛋白質之損失。藉由MBCD進行之去脂質化較佳地自IFV移除膽固醇但不移除蛋白質。此外，經MBCD處理之IFV具有更小活體外感染性，但感染性並未完全消除。用二異丙基醚及 MBCD 進行 IFV 去脂質 化 之 比較： 除MBCD之外，使用另一脂質溶劑二異丙基醚(DIPE)來對IFV進行去脂質。用不同濃度之DIPE或用50 mM MBCD處理相同起始物質之IFV樣本。在37℃下用2%、4%、8%或12% DIPE或50 mM MBCD處理相同數量IFV之等分試樣30分鐘。在以2,000 ×g 短暫離心3分鐘之後，移除可見溶劑，且將含水物質在通風櫥中放置20分鐘至乾燥。隨後，以200,000 ×g 離心樣本30分鐘，以恢復病毒粒子。IFV之活體外感染性、HA活性、蛋白質含量及膽固醇含量示於表2中。 在相同條件下，濃度高達12%之DIPE處理並不如50 mM MBCD般有效地移除膽固醇。12% DIPE自IFV移除約80%之膽固醇，而50 mM MBCD移除超過98%之膽固醇。如藉由比較PFU/mL所測定，此等兩種處理之IFV感染性與模擬處理相比分別減少32倍及2250倍。藉由兩種溶劑對膽固醇質移除為相對特異性的，因為在各種處理中蛋白含量減少小於20%。在定量血球凝集(HA)活性之能力內，50 mM MBCD以與2%至8% DIPE相同的程度影響HA活性。 表2
Figure 105144117-A0304-0002
 註：a 與模擬處理相比之百分比。用溶劑處理 45 分鐘： 除了MBCD處理持續時間自30分鐘延長至45分鐘之外，重複以上處理。結果展示於下表3中。 表3
Figure 105144117-A0304-0003
 註：a 在37℃下翻滾旋轉30分鐘。b 在37℃下翻滾旋轉45分鐘。c 與模擬處理相比之百分比。 如同30分鐘處理一樣，與模擬處理相比，用50 mM MBCD處理45分鐘移除IFV之約97%膽固醇且使得病毒呈現低於超過10,000倍的活體外感染性。相比之下，10% DIPE (30分鐘處理)在移除膽固醇方面不太有效(約80%)，並使得活體外感染性降低約120倍。MBCD對HA活性之影響不顯著，而10% DIPE顯著地降低HA活性。與50 mM MBCD減少6%蛋白質含量相比，在10% DIPE處理之後蛋白質含量減少至50%。 本文中所呈現之兩組實驗表明，50 mM MBCD選擇性地移除膽固醇且使去脂質IFV呈現較低感染性，同時維持大部分蛋白質。然而，此等處理均不能完全地消除感染性且因此不能完全地使病毒不活化，故需要進一步最佳化程序以使流感病毒完全不活化。進一步最佳化可包括(但不限於)增加MBCD處理時間及用MBCD處理病毒第二時間段。實例 2 用MBCD處理PRRSV選擇性地移除膽固醇、降低病毒感染性且維持大部分病毒蛋白質含量。試劑 ： 此實例中所使用之PRRSV病毒株為RESP (中國，Jiangsu Academy of Agriculture Sciences)。所使用之宿主細胞為Marc-145 (中國，Wuxi AppTec, Shanghai)。除了在培養Marc-145細胞時使用的DMEM (Invitrogen)之外，所使用之其他試劑與上述實例1相同。PRRSV 在 Marc-145 細胞中之增殖： 根據以下程序用PRRSV接種細胞。自在T-75燒瓶中生長之Marc-145細胞中移除培養基，在室溫下用5 mL PBS沖洗Marc-145細胞單層，並且在37℃下用1.5 mL胰蛋白酶/EDTA分離細胞。使細胞再懸浮於含有10% FBS之10 mL DMEM培養基中，且藉由在4℃下以800 rpm離心5分鐘而集結成塊。使細胞再懸浮於含有10% FBS之DMEM培養基中，並且將細胞密度調整至2.5 × 105 個細胞/毫升。將十五毫升之Marc-145細胞懸浮液接種至各T75燒瓶中，且將燒瓶在37℃以及5% CO2 下放置且培育隔夜。 當Marc-145細胞匯合超過90%時，自T-75燒瓶移除DMEM，添加含有2% FBS之5 mL EMEM維持液，且以0.1之MOI用PRRSV感染細胞。在37℃以及5% CO2 下培育燒瓶60分鐘至90分鐘，且每隔15分鐘輕輕地振盪。向各燒瓶中添加含有2% FBS之十毫升EMEM維持液，並且在37℃以及5% CO2 下培育燒瓶96小時。在達到80%之CPE時(通常96小時)，收集培養物上清液以獲得PRRSV。PRRSV 之純化 根據以下程序純化PRRSV。以3,000 rpm離心所採集之培養物上清液20分鐘以移除細胞碎屑，且採集上清液。以100,000 xg 離心該上清液60分鐘，以使PRRSV集結成塊。使PRRSV離心塊再懸浮於適當體積之生理緩衝鹽水(PBS)中至約1 × 109 PFU/mL之力價。在-80℃下儲存100 μL經濃縮PRRSV之等分試樣。 使用以下程序針對活體外感染性滴定PRRSV。使用上述細胞培養物及轉移程序，使Marc-145細胞以1.5 × 105 個細胞/毫升之最終密度懸浮於含有10% FBS之DMEM中。向6孔培養盤之各孔中添加兩毫升之Marc-145細胞懸浮液，並且在37℃以及5% CO2 下培育培養盤隔夜。在37℃水浴中使病毒樣本解凍，之後在4℃下以500 ×g 離心10分鐘。使用EMEM作為稀釋緩衝液，製備病毒之1/10稀釋系列且在使用之前在4℃下進行儲存。 當Marc-145細胞匯合至少90%時，移除培養基，且向各孔中添加0.5 mL稀釋緩衝液及0.5 mL病毒稀釋液。對於陰性對照組，使用1.0 mL稀釋緩衝液。在添加各稀釋液之後立刻輕輕振盪培養盤。在37℃下、在5% CO2 中培育培養盤60分鐘至90分鐘，且每隔15分鐘搖動培養盤。接著，在37℃下用含2.5%低熔點瓊脂糖之PBS溶液與稀釋緩衝液之3 mL的1:4混合物覆蓋各6孔培養盤之各孔。在室溫下培育培養盤15分鐘以使覆層混合物固化，且接著在37℃以及5% CO2 下培育培養盤96小時。 使用以下程序使溶菌斑顯色。在溶菌斑充分形成之後(感染96小時後)，添加1 mL 4%多聚甲醛，且在室溫下培育培養盤1小時。棄去溶解的瓊脂糖，並且向各孔中添加0.5 mL之0.5%結晶紫。在與結晶紫一起培育15分鐘之後對溶菌斑數目進行計數，且基於稀釋因數轉換為力價。PRRSV 之活體外增殖： 對於蛋白質及膽固醇分析而言，大於1 × 107 PFU/mL之PRRSV力價係較佳的。PRRSV-RESP病毒株在Marc-145細胞中之增殖產生力價＞ 1 × 108 PFU/mL之經濃縮病毒樣本。PRRSV 之去脂質化： 根據以下程序進行溶劑處理及模擬處理。在艾本德試管中將約1 × 109 PFU/mL之經濃縮PRRSV儲備液之一百微升等分試樣稀釋至900 μL PBS中，以達到1.0 - 5.0 × 108 PFU/mL之最終力價。對於溶劑處理，添加含MBCD之PBS至所需濃度，或對於模擬處理添加相同體積之PBS。隨後用封口膜封蓋且密封艾本德試管。將所有樣本固持在預加熱至37℃之SHZ-82恆溫定軌氣浴震盪器(中國，Changzhou Guohua Appliance Co.)中。在37℃下以220 rpm之定軌旋轉速度使經溶劑處理樣本及經模擬處理樣本旋轉所需時間。在微離心機中使各別樣本旋轉1分鐘，且將含有IFV之上清液轉移至超離心機試管。以100,000 ×g (OPTIMATM L-100XP；Beckman Coulter, Inc.，Indianapolis IN)離心試管30分鐘以使PRRSV集結成塊，且棄去上清液。使PRRSV離心塊再懸浮於200 µL PBS中，且在進一步分析之前在-80℃下進行儲存。去脂質 PRRSV 之表徵 ： 根據製造商的說明使用BCA分析(PIERCE™ BCA蛋白質分析套組)來測定去脂質及經模擬處理之PRRSV的蛋白質含量。亦使用根據製造商之說明進行的膽固醇分析(Amplex® Red膽固醇分析套組，Life Technologies，Grand Island，NY)來測定膽固醇含量。使用與上文所闡述相同的程序來量測活體外感染性。MBCD 之濃度依賴性： 在第一去脂質化實驗中，在37℃下用5 mM、10 mM、20 mM、30 mM及50 mM MBCD對PRRSV進行去脂質化，持續60分鐘。在超離心之後，使用上述程序，測試經模擬處理樣本及去脂質樣本在Marc-145細胞中之活體外感染性且測定蛋白質及膽固醇含量。去脂質PRRSV之感染性、蛋白質含量及膽固醇含量示於表4中。 表4
Figure 105144117-A0304-0004
 註：a 與模擬處理相比之百分比。 起始PRRSV-RESP儲備液之力價為1.0 × 108 PFU/mL，將0.1 mL之儲備液與0.9 mL PBS以及適當量之MBCD混合。在37℃下以220 RPM定軌震盪60 min之後，藉由超速離心使病毒樣本集結成塊且將其懸浮於最終體積0.2 mL之PBS中。經模擬處理樣本之力價為1.6 × 107 PFU/mL (表4)，表明在模擬處理之後病毒活性恢復超過30%。MBCD處理以很大程度上濃度依賴性方式減少感染性及膽固醇含量，而在所有MBCD濃度下總蛋白質含量恢復88%至98%(參見 表4)。與模擬樣本相比，在用50 mM MBCD處理之後，去脂質PRRSV保留約28%之起始膽固醇量，且PRRSV感染性減少約380倍，但同樣未完全消除。去脂質 PRRSV 樣本之盲傳分析 去脂質PRRSV樣本中之一者(100 mM MBCD持續90分鐘)的力價在檢測限度內。此引發樣本中是否存在任何感染性病毒粒子之問題。為解決是否存在該等感染性病毒粒子之問題，用100 mM MBCD對新樣本進行去脂質90分鐘且使該新樣本經受下述盲傳分析。雖然對照PRRSV-RESP樣本具有1.6 × 106 PFU/mL之力價，但藉由盲傳分析未自用100 mM MBCD去脂質90分鐘之PRRSV樣本檢測到溶菌斑。用100 mM MBCD去脂質90分鐘很可能完全地使PRRSV-RESP不活化。 根據以下程序進行盲傳分析。將藉由上述程序製備之一百微升去脂質PRRSV樣本轉移至含有Marc-145細胞之6孔培養盤的各孔。在37℃下在5% CO2 中培育培養盤4天。預進行三個連續冷凍-解凍循環來使細胞溶解，且收集含有PRRSV之上清液且在4℃下以800 rpm離心5 min。將四百微升上清液轉移至含Marc-145細胞之燒瓶。在37℃及5% CO2 下培育燒瓶4天。重複冷凍-解凍循環，且再次如前所述收集、離心上清液，並將其轉移至含Marc-145細胞之燒瓶。再次重複冷凍-解凍循環，再次收集且離心上清液，且含有PRRSV之上清液在進一步使用之前在-80℃下冷凍。如上所述，使用Marc-145細胞測定最終所收集樣本之PRRSV力價。實例 3 此研究之目標為經由利用甲基β環糊精(MBCD)之去脂質化方法來測定PRRSV病毒株ND 99-14之不活化的時間依賴性。自CTD, Inc. (Alachua, FL)購買呈粉末形式的MBCD。藉由向280 g MBCD中添加1000 mL水來製備300 mM儲備溶液，以產生儲備溶液。病毒去脂質 化 ： 使用在2016年2月18日申請之同在申請中的申請案US第62/296,658號中所描述的PRRSV病毒株ND99-14來進行不活化動力學實驗。一般而言，方法涉及添加最終濃度為40 mM之MBCD以及在室溫及恆定混合下培育總共72小時。MBCD之添加發生在2個步驟中，添加20 mM開始不活化過程，24小時之後添加額外20 mM MBCD且在室溫及恆定混合下培育額外48小時。室溫在約20℃與約25℃之間，且最佳在約22℃與約24℃之間。 初始地向病毒中添加MBCD溶液之300 mM儲備液 (藉由向8.5 L病毒原液中添加567 mL MBCD溶液)直至20 mM之最終濃度。在室溫及以50 rpm混合下培育病毒24小時。在添加MBCD後的0小時、15分鐘、4小時、8小時、12小時、16小時、20小時及24小時處採集30 mL病毒。各時間點之等分試樣在進一步使用之前在4℃及-70℃下進行儲存。 在採集24小時樣本之後，將病毒轉移至新的容器。添加額外20 mM MBCD (向9.75 L病毒原液中添加650 mL MBCD)以達成40 mM之最終濃度。在室溫下培育病毒額外48小時。在第一次添加MBCD之後的30、36、42、48、60、72、84及96小時處採集30 mL病毒。各時間點之等分試樣在進一步使用之前在4℃及-70℃下進行儲存。病毒量測： 向在96孔培養盤上培養之MARC145細胞中添加模擬不活化病毒及不活化病毒之十倍連續稀釋液(10- 1 至10- 9 )。在37℃及5% CO2 下培育培養盤96小時，使活病毒感染細胞。藉由觀測由於病毒感染MARC145細胞而引起之細胞病變效應(CPE)來測定病毒複製。就CPE而言將含有病毒稀釋液之各孔評分為陽性或陰性，且使用Reed-Muench TCID50 計算，將每一稀釋液之所得陽性或陰性孔數用於測定TCID50 /mL。以與處理不活化病毒相同的介質及溫度條件(不添加不活化劑)來處理模擬不活化病毒。 使用培養在T-225燒瓶上之MARC145細胞來進行模擬不活化病毒及不活化病毒以及僅對照培養基的三次連續盲傳。向MARC145細胞之2天培養物中添加不活化樣本及對照樣本(將3 mL樣本添加至27 mL培養基中)，且在37℃及5% CO2 下培育7天。目測燒瓶中是否存在CPE。就CPE而言將每一燒瓶評分為陽性(+)或陰性(-)且記錄。收集來自燒瓶之上清液，且將其添加至含有MARC-145細胞之2天培養物的一組新T-225燒瓶中，並且在37℃及5% CO2 下培育7天。重複上述程序兩次，以獲得3次連續病毒繼代之CPE觀測值。 使用在如所示之2個不同溫度下儲存的樣本來測定在96小時培育期間的所有採集時間點之PRRS病毒力價。0表明力價不可檢測或未藉由PRRS活病毒TCID50 測定分析測定。表5中所呈現之結果表明，在2步過程中添加40 mM能夠在力價為至少8 log10 TCID50 /mL的情況下使PRRS病毒不活化。較高力價表明在20 mM MBCD的情況下未完全失活，但在第二次添加MBCD使濃度升至40 mM之後，病毒係不可檢測的。該等結果亦顯示時間為不活化之較次要促成因素。在給定MBCD濃度的情況下，更長的培育時間並未提高不活化程度。在培育15分鐘之後，20 mM濃度之MBCD減少活病毒大致2.5 log，且檢測到之活病毒水準持續24小時保持不變。在培育24小時第二次添加20 mM MBCD使病毒在下一次樣本採集時間點時(30小時)不活化至不可檢測水準。在不同儲存溫度之40 mM MBCD存在下亦觀測到存活性差異。在-70℃下儲存之具有可檢測活病毒之時間點樣本比4℃下之彼等者具有至少少100倍的病毒。 表5
Figure 105144117-A0304-0005
 a Log10 TCID50 /mL實例 4 此研究之目標為在接種疫苗攻擊研究中評估實驗性不活化PRRSV疫苗之安全性及功效。基於疫苗減少肺病變及降低病毒血症水準之能力評估疫苗。所關注之疫苗特徵包括儲存溫度、劑量(1相較於2)及佐劑添加。在Veterinary Resources, Inc. (Cambridge, Iowa)之BSL-2設施中進行該研究。在愛荷華州立大學獸醫診斷實驗室(Iowa State University Veterinary Diagnostic Laboratory) (Ames, Iowa)及南達科塔州立大學獸醫診斷實驗室(South Dakota State University Veterinary Diagnostic Laboratory) (Brookings, South Dakota)處進行實驗室分析。PRRSV血清反應陰性之六十(60)隻約3週齡的臨床上健康的斷奶小豬參與該研究。按遞減的體重來排序小豬，且形成10個區塊，每個區塊6隻動物。小豬被隨機分配至六個處理組中之一組(每組10隻小豬)。處理組包括一個未疫苗接種對照組(T01)及五個疫苗接種實驗性不活化PRRSV疫苗之組。藉助使用甲基-β-環糊精(MBCD)去脂質化之手段使疫苗不活化。組T02及組T03疫苗不含佐劑，且在2℃至8℃ (T02)或-20℃ (T03)下儲存。組T04及組T05疫苗含有MONTANIDE™ Gel 01佐劑(Seppic)，且投與一次(T04)或兩次(T05)。組T06疫苗含有MONTANIDE™ IMS 1313 VG N PR佐劑(Seppic)，且給予兩次。 經由頂載式飼料槽用標準市售藥用(CTC/DENAGARD® )開口飼料(NRC，2012)隨意餵養小豬。小豬經由乳頭飲水器隨意飲用乾淨的飲用水。在接種疫苗(DPV)後第10天，用EXCEDE® 處理所有研究小豬。此外，用單次劑量之VITAL E® -500及EXCEDE® 21 DPV之另一處理來處理所有小豬。根據標籤說明投與所有藥劑。疫苗 PRRSV病毒株SD 11-21 (繼代程度84)用作當前研究之抗原。SD11-21為被調適用於在MARC-145細胞系之細胞培養物中生長經過84次繼代以及三回溶菌斑純化及蔗糖梯度離心之野生病毒株，如在2016年2月18日申請之同在申請中的申請案US第62/296,658號中所描述。在接種有連接至HILLEX II微載體(Pall Corporation, Port Washington, NY)之MARC-145的5 L BioBLU一次性生物反應器(Eppendorf, Hamburg, Germany)中，在補充有2%胎牛血清(Sigma, St. Louis, MO)及50 ug/mL健大黴素(Gentamycin) (Life Technologies, Grand Island, NY)之OPTI-MEM培養基(Life Technologies, Grand Island, NY)中產生第85次繼代(p85)。如藉由實例3中所描述之病毒滴定分析所測定，所收集之p85病毒原液的力價為7.3 log10 TCID50 。 在1 L瓶中，將19.7 mL之300 mM MBCD (Sigma, St. Louis, MO)儲備溶液添加至300 mL之SD11-21病毒原液中。在室溫及以50 rpm恆定混合下培育混合物24小時。在培育24小時之後，添加額外9.8 mL之MBCD儲備溶液，且在室溫及以50 rpm恆定混合下培育混合物額外24小時。在總共48小時之室溫培育的情況下，MBCD之最終濃度為30 mM。亦製備模擬不活化病毒原液，添加等量水代替MBCD，培育時間及混合與不活化病毒相同。模擬不活化病毒用作病毒量測分析之對照組。處理之後，模擬不活化病毒以6.5 log10 TCID50 /mL存在，而在MBCD不活化病毒溶液中未檢測到活病毒。病毒原液在進一步使用之前在4℃下進行儲存。 疫苗經調配以包括穩定劑及防腐劑。OPTI-MEM® I血清減少的培養基用作摻合稀釋劑。佐劑包括MONTANIDE™ Gel 01 (Seppic)及MONTANIDE™ 1313 VG N PR (Seppic)之市售調配物。對照組產品為磷酸鹽緩衝鹽水(PBS)之市售製劑(Corning Cellgro, Mediatech Inc.)。無佐劑之疫苗含有去脂質病毒、25%穩定劑B、作為防腐劑之25 μg/ml健大黴素及摻合稀釋劑。含佐劑之疫苗含有20%之指定佐劑。穩定劑B含有2.5% D-甘露醇(Sigma)、1.2% A型明膠(Fisher, Pittsburgh, PA)、1% NZ胺酪蛋白水解產物(Sigma)、5%蔗糖(Sigma)及pH 7.0至7.2之含6.2%海藻糖(Fisher)之超純水(Life Technologies)。接種疫苗及攻擊 參與研究之60隻小豬中之每一者在攻擊前第35天接種疫苗。在頸部右側給T01至T05中之小豬注射呈1.0 mL肌肉內劑量之指定治療劑。給T06中之小豬注射1.5 mL。T01至T03及T05以及T06中之小豬在第14天後(攻擊前第-21天)以相同方式再次接種疫苗。在0 DPC時，藉由在攻擊前立即解凍病毒株NADC-20之冷凍等分試樣來製備攻擊物質。在於具有伊格爾鹽(Earle's salts)及來自Mediatech, Inc.之L-麩醯胺酸(MEM)的伊格爾最低必需培養基(Minimum Essential Medium Eagle)中稀釋之後，力價經測定為1.26×105 . 0 TCID50 /ml。每一小豬以物理方式被限制為頭朝上，以便攻擊。使用3 mL非魯爾(luer)鎖定注射器經鼻內遞送2 mL劑量，每鼻孔大致1 mL。結果 在14 DPC時，在每一位點程序將動物人道地安樂死。切下肺臟，且由對處理方式不知情之研究調查人員評分。以目測及觸診兩種方式檢測七個肺葉中每一者之由PRRSV引起的整體特徵病變。每一肺葉中病變/固結的量經評分為肺葉之0與100%之間的實際值。將每一肺葉之得分鍵入重量公式，以計算肺病變之百分比。根據下式計算總肺病變之百分比：總肺病變之百分比 = {(0.10 × 左頂端) + (0.10 × 左心臟) + (0.25 × 左隔膜) + (0.10 × 右頂端) + (0.10 ×右心臟) + (0.25 ×右隔膜) + (0.10 ×中部)}。 此外，在進一步分析之前，使用反正弦平方根轉換總肺病變之百分比。藉由包括固定處理因素(SAS中之混合程序)及隨機區塊因素之混合線性模型來分析經轉換數據。 肺病變得分之統計學分析結果概述於表6中。主要處理作用係統計顯著的(P = 0.0003)。與對照組(T01)之比較表明Gel 01佐劑組(T04及T05)中顯著較低的(P ＜ 0.05)肺損害%。又，投與兩次且儲存在2℃至8℃下之非佐劑疫苗(T02)相比於用儲放在-20℃下之疫苗處理的類似組(T03)，產生顯著更低的(P ＜ 0.05)病變得分。 表6.     平均肺病變得分 - 經反正弦轉換之肺損害% (主要處理作用：P = 0.0003)
Figure 105144117-A0304-0006
 *在P ＜ 0.05的情況下，T01對比T04及T05顯著地不同1 未經轉換之平均值2 經反向轉換之平均值 在攻擊前第-35天及第-21天採集血液樣本以用於測定病毒血症水準。此外，在0、3、7、10及14 DPC時採集血液樣本以用於測定病毒血症水準。使用qRT-PCR技術測試樣本是否存在PRRS病毒核酸。 在非正態分佈的情況下，在分析之前將血清學及病毒血症數據轉換為log10 單位。使用重複量測混合模型(混合程序)來分析經轉換值。統計模型包括處理、時間及作為固定因素之處理×時間相互作用。區塊作為隨機因素包括於模型中。若處理×時間相互作用顯著(P ＜ 0.05)，則評估處理時間內之作用。若相互作用並不顯著，則評定主要處理作用。呈現最小均方值(經反向轉換)及標準誤差。 病毒血症水準之分析概述於表7中。在PRRSV攻擊之前未在任一小豬中觀測到病毒血症，從而確證疫苗病毒係不活化的。自統計分析排除攻擊前之時間點。在攻擊之後，處理×天數相互作用並不顯著(P = 0.0974)，因此評估主要處理作用。處理作用顯著(P = 0.0107)。與對照組(T01)之比較表明所有佐劑疫苗組(T04、T05及T06)中顯著較低的(P ＜ 0.05)水準。之前進行之對比中無一者為統計顯著的。 表7.     病毒血症 - 3、7、10及14 DPC時之PRRSV基因組複本/毫升之幾何平均值(經log10 轉換) (主要處理作用：P = 0.011；處理×天數相互作用：P = 0.097)
Figure 105144117-A0304-0007
 *在P ＜ 0.05的情況下，T01對比T04、T05、T06顯著地不同。1 未經轉換之log10 平均值2 經反向轉換之平均值結論 與對照組相比，用經一次劑量或兩次劑量之MBCD不活化的Gel 01佐劑疫苗接種小豬有效地顯著減少肺病變及病毒血症水準兩者。在用儲存在2℃至8℃下之經其他MBCD不活化之疫苗(T02及T06)接種的小豬中，肺病變在數值上減少。由於肺病變得分存在變化，故此等差異在P ＜ 0.05水準下並不顯著。含有佐劑(MONTANIDE™ Gel 01或1313)之所有MBCD疫苗顯著地減少病毒血症。在將來的研究中，當NADC-20病毒株用作攻擊物質以檢測肺病變及病毒血症兩者中有意義的生物差異時，可能需要更大的樣本容量。 儲存溫度似乎對疫苗功效具有影響。相比於儲存在2℃至8℃下之疫苗，將疫苗儲存在-20℃下對疫苗功效具有負面影響。 在疫苗接種含Gel 01佐劑之經MBCD不活化之疫苗的小豬中，在第一次接種14天之後添加額外接種疫苗未進一步改良疫苗功效。 將Gel 01佐劑包括於經MBCD不活化之疫苗中並不影響疫苗功效。 如藉由在第二次接種疫苗之後，Seppic MONTANIDE™ Gel 01佐劑組中無接種疫苗後全身性反應、無注射位點病變且僅出現短暫(1或2天)發熱反應所證實，經MBCD不活化之疫苗對生長豬類係安全的。Therefore, the present invention provides a method for preparing inactivated enveloped viruses, the method comprising: mixing a solution containing enveloped viruses with a first MBCD solution to obtain a first mixture; incubating the first mixture for a first period of time; Mixing the enveloped virus from the first mixture with a second MBCD solution to obtain a second mixture; and incubating the second mixture for a second period of time. The concentration of MBCD in the first mixture is at least 5 mM to about 100 mM. Further, the concentration of MBCD in the first mixture is about 20 mM to about 40 mM. Furthermore, the concentration of MBCD in the first mixture is about 20 mM, about 30 mM, or about 40 mM. The present invention provides a method for preparing inactivated enveloped viruses, the method comprising: mixing a solution containing enveloped viruses with a first MBCD solution to obtain a first mixture; incubating the first mixture for a first period of time; The enveloped virus of the first mixture is mixed with a second MBCD solution to obtain a second mixture; and the second mixture is incubated for a second period of time; wherein the concentration of MBCD in the second mixture is at least 10 mM to about 100 mM. Further, the concentration of MBCD in the second mixture is about 30 mM to about 50 mM. Furthermore, the concentration of MBCD in the second mixture is about 30 mM, about 40 mM, or about 50 mM. The present invention provides a method for preparing inactivated enveloped viruses, the method comprising: mixing a solution containing enveloped viruses with a first MBCD solution to obtain a first mixture; incubating the first mixture for a first period of time; The enveloped virus of the first mixture is mixed with a second MBCD solution to obtain a second mixture; and the second mixture is incubated for a second period of time; wherein the first period of time is about 15 minutes to about 24 hours. Furthermore, the first time period is about 4 hours to about 24 hours. Furthermore, the first time period is about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours. The present invention provides a method for preparing inactivated enveloped viruses, the method comprising: mixing a solution containing enveloped viruses with a first MBCD solution to obtain a first mixture; incubating the first mixture for a first period of time; The enveloped virus of the first mixture is mixed with a second MBCD solution to obtain a second mixture; and the second mixture is incubated for a second period of time; wherein the second period of time is about 4 hours to about 48 hours. Furthermore, the second time period is about 24 hours to about 48 hours. Furthermore, the second time period is about 24, 30, 36, 40, 44, or 48 hours. The present invention provides a method for preparing inactivated enveloped viruses, the method comprising: mixing a solution containing enveloped viruses with a first MBCD solution to obtain a first mixture; incubating the first mixture for a first period of time; The enveloped virus of the first mixture is mixed with a second MBCD solution to obtain a second mixture; and the second mixture is incubated for a second period of time; wherein the temperature of the first mixture during the first period of time is room temperature or about 20°C to about 25°C. Furthermore, the temperature of the first mixture during the first time period is about 22°C to about 24°C. The present invention provides a method for preparing inactivated enveloped viruses, the method comprising: mixing a solution containing enveloped viruses with a first MBCD solution to obtain a first mixture; incubating the first mixture for a first period of time; The enveloped virus of the first mixture is mixed with a second MBCD solution to obtain a second mixture; and the second mixture is incubated for a second period of time; wherein the temperature of the second mixture during the second period of time is room temperature or about 20°C to about 25°C. Furthermore, the temperature of the second mixture during the second time period is about 22°C to about 24°C. The present invention provides a method for preparing inactivated enveloped viruses, the method comprising: mixing a solution containing enveloped viruses with a first MBCD solution to obtain a first mixture; incubating the first mixture for a first period of time; The enveloped virus of the first mixture is mixed with a second MBCD solution to obtain a second mixture; and the second mixture is incubated for a second period of time; wherein the enveloped virus and the first mixture are obtained before the step of obtaining the second mixture One mixture separates. Alternatively and without limitation, the second MBCD solution can be directly mixed with the first mixture. The direct mixing can be performed in the same inactivated container as the first mixing, or the first mixture can be moved to a new inactivated container before mixing with the second MBCD solution. The present invention provides a method for preparing inactivated enveloped viruses, the method comprising: mixing a solution containing enveloped viruses with a first MBCD solution to obtain a first mixture; incubating the first mixture for a first period of time; The enveloped virus of the first mixture is mixed with a second MBCD solution to obtain a second mixture; and the second mixture is incubated for a second period of time; wherein the first period of time further comprises mixing the first mixture. The stirring can be about 30 rpm to about 100 rpm. Preferably, the stirring may be about 40 rpm to about 60 rpm. Optimally, the stirring may be about 50 rpm. The present invention provides a method for preparing inactivated enveloped viruses, the method comprising: mixing a solution containing enveloped viruses with a first MBCD solution to obtain a first mixture; incubating the first mixture for a first period of time; Mixing the enveloped virus of the first mixture with a second MBCD solution to obtain a second mixture; and incubating the second mixture for a second period of time; wherein the second period of time further comprises mixing the second mixture. The stirring can be about 30 rpm to about 100 rpm. Preferably, the stirring may be about 50 rpm. The present invention provides a method for preparing inactivated enveloped viruses, the method comprising: mixing a solution containing enveloped viruses with a first MBCD solution to obtain a first mixture; incubating the first mixture for a first period of time; The enveloped virus of the first mixture is mixed with a second MBCD solution to obtain a second mixture; and the second mixture is cultivated for a second period of time; wherein the enveloped virus is a porcine reproductive and respiratory syndrome (PRRS) virus. The enveloped virus may also be porcine epidemic diarrhea virus (PEDV). The present invention provides a lipid-free enveloped virus, which is obtained by the following method: mixing a solution containing an enveloped virus with a first MBCD solution to obtain a first mixture; incubating the first mixture for a first period of time; A mixture of the enveloped virus is mixed with a second MBCD solution to obtain a second mixture; and the second mixture is incubated for a second period of time. Preferably, the concentration of MBCD in the first mixture is at least 5 mM to about 100 mM, or about 20 mM to about 40 mM. Preferably, the concentration of MBCD in the second mixture is at least 10 mM to about 100 mM, or about 30 mM to about 50 mM, or about 30 mM, about 40 mM, or about 50 mM. Preferably, the first time period is about 15 minutes to about 24 hours, or about 4 hours to about 24 hours, or about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 Hour. Preferably, the second time period is about 4 hours to about 48 hours, or about 24 hours to about 48 hours, or about 24, 30, 36, 40, 44, or 48 hours. Preferably, the temperature of the first mixture during the first time period is room temperature, or about 20°C to about 25°C, or even about 22°C to about 24°C. Preferably, the temperature of the second mixture during the second time period is room temperature, or about 20°C to about 25°C, or even about 22°C to about 24°C. Preferably, the enveloped virus is separated from the first mixture before the step of obtaining the second mixture. Alternatively and without limitation, the second MBCD solution can be directly mixed with the first mixture. The direct mixing can be performed in the same inactivated container as the first mixing, or the first mixture can be moved to a new inactivated container before mixing with the second MBCD solution. Preferably, the first time period further comprises mixing the first mixture, wherein the stirring may be about 30 rpm to about 100 rpm, about 40 rpm to about 60 rpm, or about 50 rpm. Preferably, the second time period further comprises mixing the second mixture, wherein the stirring may be about 30 rpm to about 100 rpm, about 40 rpm to about 60 rpm, or about 50 rpm. Preferably, the enveloped virus is porcine reproductive and respiratory syndrome (PRRS) virus or porcine epidemic dysentery virus (PEDV). The present invention provides a vaccine containing a lipid-free enveloped virus, which is obtained by the following method: mixing a solution containing the enveloped virus with a first MBCD solution to obtain a first mixture; cultivating the first mixture For a period of time; mixing the enveloped virus from the first mixture with a second MBCD solution to obtain a second mixture; and incubating the second mixture for a second period of time. Preferably, the concentration of MBCD in the first mixture is at least 5 mM to about 100 mM, or about 20 mM to about 40 mM. Preferably, the concentration of MBCD in the second mixture is at least 10 mM to about 100 mM, or about 30 mM to about 50 mM, or about 30 mM, about 40 mM, or about 50 mM. Preferably, the first time period is about 15 minutes to about 24 hours, or about 4 hours to about 24 hours, or about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 Hour. Preferably, the second time period is about 4 hours to about 48 hours, or about 24 hours to about 48 hours, or about 24, 30, 36, 40, 44, or 48 hours. Preferably, the temperature of the first mixture during the first time period is room temperature, or about 20°C to about 25°C, or even about 22°C to about 24°C. Preferably, the temperature of the second mixture during the second time period is room temperature, or about 20°C to about 25°C, or even about 22°C to about 24°C. Preferably, the enveloped virus is separated from the first mixture before the step of obtaining the second mixture. Alternatively and without limitation, the second MBCD solution can be directly mixed with the first mixture. The direct mixing can be performed in the same inactivated container as the first mixing, or the first mixture can be moved to a new inactivated container before mixing with the second MBCD solution. Preferably, the first time period further comprises mixing the first mixture, wherein the stirring may be about 30 rpm to about 100 rpm, about 40 rpm to about 60 rpm, or about 50 rpm. Preferably, the second time period further comprises mixing the second mixture, wherein the stirring may be about 30 rpm to about 100 rpm, or about 50 rpm. Preferably, the enveloped virus is porcine reproductive and respiratory syndrome (PRRS) virus or porcine epidemic dysentery virus (PEDV). The present invention provides a vaccine containing a lipid-free enveloped virus, which is obtained by: mixing a solution containing an enveloped virus with a first MBCD solution to obtain a first mixture; cultivating the first mixture For a period of time; mixing the enveloped virus from the first mixture with a second MBCD solution to obtain a second mixture; and cultivating the second mixture for a second period of time; wherein the vaccine further comprises an adjuvant, a stabilizer, and a preservative At least one of an agent and a blended diluent. Preferably, the adjuvant is an aqueous polymer adjuvant, wherein the polymer is acrylate or polyacrylate. Preferably, the stabilizer includes at least one of sugar, carbohydrate, protein, and gelatin. Preferably, the preservative is an antibiotic or a biologically stable compound that delays, inhibits or hinders the growth, metabolic activity or proliferation of microorganisms. Preferably, the blended diluent is water, phosphate buffered saline, cell culture medium or other solutions containing physiological salinity and pH. The present invention provides a use of a lipid-free enveloped virus, which is obtained by the following method: mixing a solution containing the enveloped virus with a first MBCD solution to obtain a first mixture; cultivating the first mixture first Time period; mixing the enveloped virus from the first mixture with a second MBCD solution to obtain a second mixture; and cultivating the second mixture for a second time period; wherein the use includes treating diseases caused by enveloped viruses or symptom. The present invention provides a use of a lipid-free enveloped virus, which is used to manufacture a medicament for treating diseases or symptoms caused by an enveloped virus; wherein the lipid-free enveloped virus is obtained by the following method: containing an enveloped virus The solution is mixed with the first MBCD solution to obtain a first mixture; the first mixture is incubated for a first period of time; the enveloped virus from the first mixture is mixed with the second MBCD solution to obtain a second mixture; and the The second mixture for the second time period. As used herein, an "enveloped virus" is any virus that has a lipid bilayer membrane or envelope surrounding the core of a nucleoprotein. The viral envelope is usually derived from the host cell membrane and contains phospholipids, glycolipids, sphingolipids, and sterols (such as cholesterol). Enveloped viruses include viruses that have a genome encoded by DNA or RNA. Types of enveloped viruses include (but are not limited to): herpes virus, pox virus, iridovirus, hepatitis virus, retrovirus, orthomyxovirus, arenavirus, bunyaviruses, paramucus Viruses, single-stranded anti-strand virus, baculovirus, filovirus, coronavirus, arteritis virus, flavivirus and togavirus. New enveloped viruses are constantly being discovered, and enveloped viruses are classified and reclassified as each virus is better characterized. A more complete description of enveloped viruses can be found in such texts as Fields Virology (eds by D.M. Knipe and P.M. Howley, Lippincott Williams & Wilkins, Philadelphia, PA, USA, 2013) (now its sixth edition). The term "virus" as used herein can mean a virus type or interchangeably means an individual infectious unit or a unit containing nucleic acid, protein, and envelope. "Virus" is an individual infectious unit, and this term is synonymous with the term "virion". "Delipidation" is the process of removing or removing lipids from the virus envelope. Delipidation was measured by the amount of cholesterol removed from the virus particles by MBCD. However, MBCD can also extract, remove or remove other lipids, such as phospholipids. Although the process described herein minimizes the protein loss of viral proteins due to MBCD-mediated delipidation, it also shows that MBCD interacts with some proteins. "Infectivity" refers to the ability of a virus to produce infection in a host cell, which is any cell from a human or non-human animal that can support virus replication. The infectivity can be measured, for example, by the number or percentage of host cells infected with a certain number of virus particles, or by the number or percentage of virus particles required to infect the host cells. Virus particles can be counted in an operable manner, such as by determining the number of plaque forming units (PFU) in a certain volume of virus particles. Virus particles can also be measured physically, such as by the presence of a specific viral protein detected, by enzyme-linked immunosorbent assay (ELISA), or by measuring the number of copies of the viral genome present in the solution ( For example, by real-time quantitative polymerase chain reaction (qPCR) detection). An "antigen" is any molecule that can be specifically detected by an organism's immune system. Typically, the viral antigen is a viral protein encoded by the viral genome. The presence of viral antigens can be specifically detected by both T lymphocytes and B lymphocytes. "Immunogenicity" refers to the ability of an antigen to induce an immune response. For vaccines, the immunogenicity of viral antigens will preferably cause protective immunity in animals that will reduce, alleviate or alleviate viral infections. Compared with antigens, "adjuvants" are non-specific stimulators of immune response. Adjuvants can stimulate the innate immune response by binding to and activating the pattern recognition receptor (PRR). For example, the PRR stimulants can be viruses or bacterial nucleic acids, lipids from bacteria or parasites, or bacterial proteins or toxins, or any artificially constructed mimics of these molecules. Adjuvants also include (but are not limited to): inorganic compounds that accumulate antigens to promote recognition by B lymphocytes or absorption by phagocytes, such as alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide or ammonium sulfate; oil; and detergent. Adjuvants can also be host mediators of immune signal transduction, such as (but not limited to) cytokines, lymphoid hormones, chemokines, interferons, anaphylactoxins, growth factors, differentiation factors, and adhesion molecules. As used herein, the term "treating/to treat/treatment" includes inhibiting, delaying, terminating, alleviating, slowing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Treatment can be applied prophylactically or therapeutically. As used herein, "administration to animals" includes transdermal, subcutaneous, intramuscular, transmucosal, submucosal, transdermal, oral, or intranasal administration. Administration can include injection or topical administration. The following experimental examples illustrate the delipidation process. It should be understood that other embodiments and uses will be obvious to those skilled in the art, and the present invention is not limited to these specific illustrative examples or preferred embodiments. For example (but not limited to) depending on the type and type of enveloped virus and the host cell in which the enveloped virus replicates, the enveloped virus contains different levels of cholesterol in its envelope. Those familiar with this technology will understand that the delipidation process described in this article may need to be optimized for each specific virus stock. The membrane of microorganisms may contain cholesterol, hopanes or other sterols and steroid molecules, and therefore pathogens that are not viruses can also be inactivated by the methods disclosed herein.Instance 1 Use the following procedure to delipidate the human influenza virus H1N1 A/WSN/33 strain. In the following example, delipidation of influenza virus (IFV) weakens infectivity. Since the delipidization method of the present invention preserves the viral envelope proteins, it is expected that animals vaccinated with delipidated IFV will produce an immunogenic response that protects them from pathogenic doses of virulent virus infection.Reagent : The human influenza virus H1N1 A/WSN/33 strain and its host cell strain MDCK can be purchased from Wuxi AppTec (Shanghai, China). Other reagents include MEM (Invitrogen, Carlsbad, CA); EMEM (Sigma-Aldrich, St. Louis, MO); ULTRAMDCK™ serum-free medium (Lonza, Inc., Allendale, NJ); Fetal Bovine Serum (FBS; Invitrogen); 0.25% Trypsin-EDTA (ethylenediaminetetraacetic acid; Invitrogen); methyl-β-cyclodextrin (MBCD, Sigma-Aldrich); AMPLEX Red cholesterol analysis kit (Invitrogen); PIERCE™ BCA protein analysis kit ( Rockford, IL); and MTT (Sigma-Aldrich).H1N1 A / WSN / 33 Influenza virus in MDCK In cell line proliferation: Cells were seeded with IFV according to the following procedure. The medium was removed from MDCK cells grown in T-75 flasks, the MDCK cell monolayer was washed with 5 mL PBS at room temperature, and the cells were detached with 1.5 mL trypsin/EDTA at 37°C. The cells were resuspended in 10 mL MEM containing 10% FBS, and agglomerated into clumps by centrifugation at 800 rpm at 4°C for 5 minutes. Resuspend the cells in MEM containing 10% FBS and adjust the cell density to 2.5 × 105 Cells/ml. Fifteen milliliters of MDCK cell suspension was inoculated into each T-75 flask, and the flask was kept at 37°C and 5% CO2 Place under and incubate overnight. When MDCK cells are more than 90% confluent, remove the MEM from the T75 flask, add 5 mL EMEM maintenance solution containing 1% FBS and 1 μg/mL trypsin, and infect the cells with influenza virus at a rate of infection (MOI) of 0.01. At 37℃ and 5% CO2 Incubate the flask for 60 to 90 minutes, shaking gently every 15 minutes. Add 10 ml of EMEM maintenance solution containing 1% FBS and 1 μg/mL trypsin to each flask, and keep it at 37°C and 5% CO2 Incubate the flask for 48 hours. When approximately 80% cytopathic effect (CPE) of the virus is achieved (usually after about 48 hours), the culture supernatant is collected to obtain IFV.H1N1 A / WSN / 33 IFV Of Purification and titration : IFV was purified according to the following procedure. The collected culture supernatant was centrifuged at 3,000 rpm for 20 minutes to remove cell debris, and the supernatant was collected. The supernatant was centrifuged at 38,000 rpm for 60 minutes to agglomerate the IFV. Resuspend the IFV centrifugal block in an appropriate volume of physiological buffered saline (PBS) to obtain >1.0 × 109 The final power value of plaque forming unit/mL (PFU/mL). Store 100 μL aliquots of this concentrated IFV stock solution at -80°C. The following procedure was used to titrate IFV for in vitro infectivity. Use the above cell culture and transfer procedure to 2.5 × 105 At a final density of 1 cell/ml, MDCK cells were suspended in MEM containing 10% FBS. Add 2 ml of MDCK cell suspension to each well of the 6-well culture plate, and incubate at 37°C and 5% CO2 Incubate the culture plate overnight. Thaw the virus stock solution in a 37°C water bath, and then defrost it at 4°C at 500 ×g Centrifuge for 10 minutes. Using ULTRAMDCK™ serum-free medium and 2.5 μg/mL trypsin as the dilution buffer, prepare a 1/10 dilution series of the stock solution and store it at 4°C before use. When the cells are at least 90% confluent, remove the medium and add 0.5 mL dilution buffer and 0.5 mL virus dilution to each well. For the negative control group, 1.0 mL of dilution buffer was used. Gently shake the culture plate immediately after adding each dilution. At 37℃, in 5% CO2 Incubate the plate in the medium for 60 to 90 minutes, and shake the plate every 15 minutes. Next, cover each well of each 6-well culture plate with 3 mL of a 1:4 mixture of PBS solution containing 2.5% low-melting agarose and dilution buffer at 37°C. Incubate the culture plate at room temperature for 15 minutes to solidify the coating mixture; then at 37°C and 5% CO2 Incubate the culture plate for 3 days. Use the following procedure to develop plaques. After the plaque was fully formed (3 days after infection), 1 mL of 4% paraformaldehyde was added, and the culture plate was incubated at room temperature for 1 hour. Discard the dissolved agarose, and add 0.1 mL of 0.5% crystal violet to each well. After 15 minutes of incubation, the number of plaques was counted, and converted into power based on the dilution factor.Lipid removal change : The delipidation procedure of purified IFV was performed in two parallel reactions: solvent treatment, which involves contacting IFV with MBCD; and simulation treatment, in which IFV was treated in the same way but not exposed to MBCD. The IFV of the same power price is used in both the simulation treatment and the solvent treatment. The IFV in the simulated treatment can be titrated to obtain an indirect measurement of the amount of lipid-free IFV remaining after the solvent treatment. Perform solvent treatment and simulation treatment according to the following procedures. Place an aliquot (100 μL) of the IFV stock solution prepared above in an Eppendorf test tube (>1.0 × 109 PFU/mL) diluted to 900 μL PBS to achieve 1.0-5.0 × 108 The final power price of PFU/mL. For solvent treatment, add MBCD-containing PBS to a final concentration of 50 mM, or add the same volume of PBS to the simulation treatment. Then cap with parafilm and seal the Eppendorf test tube. All samples were held in an SHZ-82 constant temperature orbit determination gas bath shaker (Changzhou Guohua Appliance Co., China) preheated to 37°C. The solvent-treated sample and the simulated-treated sample were rotated at an orbital rotation speed of 220 rpm at 37°C for 30 minutes or 45 minutes. Rotate the sample for 1 minute in a microcentrifuge, and transfer the supernatant containing IFV to an ultracentrifuge test tube. With 200,000 ×g (OPTIMATM L-100XP; Beckman Coulter, Inc., Indianapolis IN) centrifuge the test tube for 30 minutes to agglomerate the IFV, and discard the supernatant. The IFV centrifuge block was resuspended in 1/10 of the initial volume added to each Eppendorf test tube above, and stored at -80°C before further analysis.Lipid removal IFV Characterization: Use BCA analysis (PIERCE™ BCA Protein Analysis Kit) according to the manufacturer's instructions to determine the protein content of lipid-free and simulated IFV. Cholesterol analysis (AMPLEX® Red Cholesterol Analysis Kit, Life Technologies, Grand Island, NY) was used to determine cholesterol content according to the manufacturer's instructions. The same titration procedure as described above was used to measure the infectivity in vitro. The hemagglutination (HA) activity is determined as follows. The freshly separated chicken blood was washed three times with PBS, and red blood cells (RBC) were resuspended in 1% PBS. Prepare virus dilutions (50 μL) in PBS, and mix these dilutions with 50 μL of RBC suspension. The mixture was added to individual wells in the 96-well culture plate, and the RBC was allowed to stand for 45 minutes. If there are RBC spots or aggregates, the well is judged to be HA negative (that is, no RBC agglutination), and if there is a smooth RBC suspension, the well is judged to be positive. Prepare two batches of delipidated and simulated IFV samples according to the procedure described above. The IFV stock solution used for testing has 2.2 × 1010 The price of PFU/mL. In the first batch ("Formulation A"), the delipidization time was 30 minutes; in the second batch ("Formulation B"), the delipidation time was 45 minutes. After ultracentrifugation, characterize the delipidated and simulated processed samples. In vitro infectivity display: The PFU of the lipid-free sample is about 1/7,000 of the PFU of the simulated sample. The infectivity results are shown in Table 1 below. Despite the difference in power and price, the protein content of the various samples is within 10% of each other. The protein content of lipid-free IFV from formulation A was 97% of the simulated sample; and the protein content of lipid-free IFV from formulation B was 93% of the simulated sample (See Table 1). It was also observed that after treatment, approximately 20% of the starting material recovered, as determined by the protein content. The recovery percentage based on protein content can be compared with the starting material (2.2 × 109 PFU/mL) and simulated processed samples (4.0 × 108 PFU/mL, which is 18% of the initial power price). Compared with the simulated treatment, less than 2% of cholesterol is retained after delipidation (See Table 1). Table 1
Figure 105144117-A0304-0001
 Note:a Infectivity score compared with simulated treatment;b The percentage compared to the simulated treatment. Simulation processing shows that physical processing (oscillation and centrifugation) causes the loss of power and protein. Delipidation by MBCD preferably removes cholesterol from IFV but not protein. In addition, IFV treated with MBCD has less infectivity in vitro, but the infectivity has not been completely eliminated.Use diisopropyl ether and MBCD conduct IFV Lipid removal change Of compare: In addition to MBCD, another lipid solvent, diisopropyl ether (DIPE), was used to delipidize IFV. Treat IFV samples of the same starting material with different concentrations of DIPE or 50 mM MBCD. Treat an aliquot of the same amount of IFV with 2%, 4%, 8% or 12% DIPE or 50 mM MBCD at 37°C for 30 minutes. At 2,000 ×g After a short centrifugation for 3 minutes, the visible solvent was removed, and the water-containing material was placed in a fume hood for 20 minutes to dry. Subsequently, with 200,000 ×g Centrifuge the sample for 30 minutes to recover virus particles. The in vitro infectivity, HA activity, protein content and cholesterol content of IFV are shown in Table 2. Under the same conditions, DIPE treatment with a concentration of up to 12% is not as effective in removing cholesterol as 50 mM MBCD. 12% DIPE removes approximately 80% of cholesterol from IFV, and 50 mM MBCD removes over 98% of cholesterol. As determined by comparing PFU/mL, the IFV infectivity of these two treatments was reduced by 32 times and 2250 times compared with the simulated treatment, respectively. The removal of cholesterol by the two solvents is relatively specific, because the protein content is reduced by less than 20% in various treatments. Within the ability to quantify HA activity, 50 mM MBCD affects HA activity to the same extent as 2% to 8% DIPE. Table 2
Figure 105144117-A0304-0002
 Note:a The percentage compared to the simulated treatment.Treat with solvent 45 minute: The above treatment was repeated except that the duration of MBCD treatment was extended from 30 minutes to 45 minutes. The results are shown in Table 3 below. table 3
Figure 105144117-A0304-0003
 Note:a Tumble and rotate for 30 minutes at 37°C.b Tumble and rotate for 45 minutes at 37°C.c The percentage compared to the simulated treatment. As with the 30-minute treatment, compared with the simulated treatment, the 50 mM MBCD treatment for 45 minutes removed about 97% of the cholesterol of IFV and rendered the virus more than 10,000 times less infectivity in vitro. In contrast, 10% DIPE (30-minute treatment) is less effective in removing cholesterol (about 80%), and reduces in vitro infectivity by about 120 times. MBCD has no significant effect on HA activity, while 10% DIPE significantly reduces HA activity. Compared with the 6% protein content reduction of 50 mM MBCD, the protein content was reduced to 50% after 10% DIPE treatment. The two sets of experiments presented in this article show that 50 mM MBCD selectively removes cholesterol and renders lipid-free IFV less infectious, while maintaining most of the protein. However, none of these treatments can completely eliminate the infectivity and therefore cannot completely inactivate the virus. Therefore, further optimization procedures are required to completely inactivate the influenza virus. Further optimization may include (but is not limited to) increasing the MBCD processing time and using MBCD to treat the virus for a second period of time.Instance 2 Treatment of PRRSV with MBCD selectively removes cholesterol, reduces viral infectivity and maintains most of the viral protein content.Reagent : The PRRSV virus strain used in this example is RESP (Jiangsu Academy of Agriculture Sciences, China). The host cell used was Marc-145 (Wuxi AppTec, Shanghai, China). Except for DMEM (Invitrogen) used when culturing Marc-145 cells, the other reagents used are the same as in Example 1 above.PRRSV exist Marc-145 Proliferation in cells: Cells were inoculated with PRRSV according to the following procedure. The medium was removed from Marc-145 cells grown in T-75 flasks, the Marc-145 cell monolayer was washed with 5 mL PBS at room temperature, and the cells were detached with 1.5 mL trypsin/EDTA at 37°C. The cells were resuspended in 10 mL DMEM medium containing 10% FBS, and agglomerated into clumps by centrifugation at 800 rpm at 4°C for 5 minutes. Resuspend the cells in DMEM medium containing 10% FBS, and adjust the cell density to 2.5 × 105 Cells/ml. Fifteen milliliters of Marc-145 cell suspension was inoculated into each T75 flask, and the flask was kept at 37°C and 5% CO2 Place under and incubate overnight. When Marc-145 cells are more than 90% confluent, remove DMEM from the T-75 flask, add 5 mL EMEM maintenance solution containing 2% FBS, and infect the cells with PRRSV at an MOI of 0.1. At 37℃ and 5% CO2 Incubate the flask for 60 to 90 minutes, shaking gently every 15 minutes. Add ten milliliters of EMEM maintenance solution containing 2% FBS to each flask, and keep it at 37°C and 5% CO2 The flask was incubated for 96 hours. When the CPE reached 80% (usually 96 hours), the culture supernatant was collected to obtain PRRSV.PRRSV Purification Purify PRRSV according to the following procedure. The collected culture supernatant was centrifuged at 3,000 rpm for 20 minutes to remove cell debris, and the supernatant was collected. Take 100,000 xg The supernatant was centrifuged for 60 minutes to agglomerate PRRSV. Resuspend the PRRSV centrifugal block in an appropriate volume of physiological buffered saline (PBS) to approximately 1 × 109 The price of PFU/mL. Store 100 μL aliquots of concentrated PRRSV at -80°C. The following procedure was used to titrate PRRSV for in vitro infectivity. Using the above cell culture and transfer procedures, make Marc-145 cells 1.5 × 105 The final density of cells/ml is suspended in DMEM containing 10% FBS. Add two milliliters of Marc-145 cell suspension to each well of the 6-well culture plate, and incubate at 37°C and 5% CO2 Incubate the culture plate overnight. Thaw the virus sample in a 37°C water bath, and then defrost it at 4°C at 500 ×g Centrifuge for 10 minutes. Using EMEM as the dilution buffer, prepare a 1/10 dilution series of the virus and store it at 4°C before use. When Marc-145 cells are at least 90% confluent, remove the medium and add 0.5 mL dilution buffer and 0.5 mL virus dilution to each well. For the negative control group, 1.0 mL of dilution buffer was used. Gently shake the culture plate immediately after adding each dilution. At 37℃, in 5% CO2 Incubate the plate in the medium for 60 to 90 minutes, and shake the plate every 15 minutes. Next, cover each well of each 6-well culture plate with 3 mL of a 1:4 mixture of PBS solution containing 2.5% low-melting agarose and dilution buffer at 37°C. Incubate the culture plate at room temperature for 15 minutes to solidify the coating mixture, and then at 37°C and 5% CO2 Incubate the culture plate for 96 hours. Use the following procedure to develop plaques. After the plaque was fully formed (96 hours after infection), 1 mL of 4% paraformaldehyde was added, and the culture plate was incubated at room temperature for 1 hour. Discard the dissolved agarose, and add 0.5 mL of 0.5% crystal violet to each well. After 15 minutes of incubation with crystal violet, the number of plaques was counted and converted to power value based on the dilution factor.PRRSV Proliferation in vitro: For protein and cholesterol analysis, greater than 1 × 107 The PRRSV power price of PFU/mL is better. The proliferation of PRRSV-RESP virus strain in Marc-145 cells produces power> 1 × 108 Concentrated virus sample of PFU/mL.PRRSV Delipidation: Perform solvent treatment and simulation treatment according to the following procedures. Put approximately 1 × 10 in an Eppendorf test tube9 A one-hundred microliter aliquot of the PFU/mL concentrated PRRSV stock solution is diluted into 900 μL PBS to reach 1.0-5.0 × 108 The final power price of PFU/mL. For solvent treatment, add MBCD-containing PBS to the desired concentration, or for simulation treatment, add the same volume of PBS. Then cap with parafilm and seal the Eppendorf test tube. All samples were held in an SHZ-82 constant temperature orbit determination gas bath shaker (Changzhou Guohua Appliance Co., China) preheated to 37°C. The time required to rotate the solvent-treated sample and the simulated-treated sample at an orbital rotation speed of 220 rpm at 37°C. Spin each sample for 1 minute in a microcentrifuge, and transfer the supernatant containing IFV to an ultracentrifuge test tube. With 100,000 ×g (OPTIMATM L-100XP; Beckman Coulter, Inc., Indianapolis IN) centrifuge the test tube for 30 minutes to agglomerate PRRSV, and discard the supernatant. The PRRSV centrifuge block was resuspended in 200 µL PBS and stored at -80°C before further analysis.Lipid removal PRRSV Representation of : Use BCA analysis (PIERCE™ BCA Protein Analysis Kit) according to the manufacturer's instructions to determine the protein content of delipidated and simulated PRRSV. Cholesterol analysis (Amplex® Red Cholesterol Analysis Kit, Life Technologies, Grand Island, NY) according to the manufacturer's instructions was also used to determine cholesterol content. The same procedure as described above was used to measure the infectivity in vitro.MBCD The concentration dependence: In the first delipidation experiment, PRRSV was delipidated with 5 mM, 10 mM, 20 mM, 30 mM and 50 mM MBCD at 37°C for 60 minutes. After ultracentrifugation, the above procedure was used to test the in vitro infectivity of the simulated processed sample and the delipidated sample in Marc-145 cells and to determine the protein and cholesterol content. The infectivity, protein content and cholesterol content of lipid-free PRRSV are shown in Table 4. Table 4
Figure 105144117-A0304-0004
 Note:a The percentage compared to the simulated treatment. The initial price of PRRSV-RESP stock solution is 1.0 × 108 PFU/mL, mix 0.1 mL stock solution with 0.9 mL PBS and appropriate amount of MBCD. After orbital shaking at 220 RPM at 37°C for 60 min, the virus samples were aggregated into clumps by ultracentrifugation and suspended in a final volume of 0.2 mL of PBS. The power price of the simulated processed sample is 1.6 × 107 PFU/mL (Table 4), indicating that the virus activity recovered more than 30% after the simulation treatment. MBCD treatment reduced infectivity and cholesterol content in a largely concentration-dependent manner, and the total protein content recovered 88% to 98% at all MBCD concentrations (See Table 4). Compared with the simulated sample, after treatment with 50 mM MBCD, lipid-free PRRSV retains about 28% of the initial amount of cholesterol, and the infectivity of PRRSV is reduced by about 380 times, but it is also not completely eliminated.Lipid removal PRRSV Blind transmission analysis of samples The power value of one of the lipid-free PRRSV samples (100 mM MBCD for 90 minutes) was within the detection limit. This raises the question of whether there are any infectious virus particles in the sample. In order to solve the problem of the presence of these infectious virus particles, a new sample was delipidated with 100 mM MBCD for 90 minutes and the new sample was subjected to the following blind analysis. Although the reference PRRSV-RESP sample has 1.6 × 106 The price of PFU/mL, but the plaque was detected by blind transmission analysis of PRRSV samples that were not delipidated with 100 mM MBCD for 90 minutes. Delipidation with 100 mM MBCD for 90 minutes is likely to completely inactivate PRRSV-RESP. Perform blind transmission analysis according to the following procedure. Transfer one hundred microliters of lipid-free PRRSV sample prepared by the above procedure to each well of a 6-well culture plate containing Marc-145 cells. At 37℃ in 5% CO2 Incubate the culture plate for 4 days. Three consecutive freeze-thaw cycles were preliminarily performed to lyse the cells, and the supernatant containing PRRSV was collected and centrifuged at 800 rpm at 4°C for 5 min. Four hundred microliters of supernatant was transferred to a flask containing Marc-145 cells. At 37°C and 5% CO2 Incubate the flask for 4 days. The freeze-thaw cycle was repeated, and the supernatant was collected and centrifuged again as described above, and transferred to a flask containing Marc-145 cells. The freeze-thaw cycle was repeated again, the supernatant was collected and centrifuged again, and the supernatant containing PRRSV was frozen at -80°C before further use. As mentioned above, Marc-145 cells were used to determine the PRRSV valence of the final collected samples.Instance 3 The goal of this study is to determine the time dependence of the inactivation of the PRRSV strain ND 99-14 through the delipidation method using methyl β cyclodextrin (MBCD). Purchase MBCD in powder form from CTD, Inc. (Alachua, FL). Prepare a 300 mM stock solution by adding 1000 mL of water to 280 g MBCD to produce a stock solution.Virus delipidation change : The PRRSV virus strain ND99-14 described in the same-in-application application US 62/296,658 filed on February 18, 2016 was used for inactivation kinetics experiments. In general, the method involves adding MBCD at a final concentration of 40 mM and incubating for a total of 72 hours at room temperature and constant mixing. The addition of MBCD takes place in 2 steps, adding 20 mM to start the inactivation process, adding an additional 20 mM MBCD after 24 hours and incubating for an additional 48 hours at room temperature and constant mixing. The room temperature is between about 20°C and about 25°C, and most preferably between about 22°C and about 24°C. Initially add 300 mM stock solution of MBCD solution to the virus (by adding 567 mL MBCD solution to 8.5 L virus stock solution) to a final concentration of 20 mM. Incubate the virus for 24 hours at room temperature with mixing at 50 rpm. 30 mL of virus was collected at 0 hour, 15 minutes, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, and 24 hours after adding MBCD. The aliquots at each time point were stored at 4°C and -70°C before further use. After 24 hours of sample collection, the virus was transferred to a new container. Add an additional 20 mM MBCD (650 mL MBCD to 9.75 L of virus stock) to achieve a final concentration of 40 mM. Incubate the virus at room temperature for an additional 48 hours. 30 mL of virus was collected 30, 36, 42, 48, 60, 72, 84, and 96 hours after the first addition of MBCD. The aliquots at each time point were stored at 4°C and -70°C before further use.Virus measurement: To MARC145 cells cultured on 96-well culture plates, add simulated inactivated virus and ten-fold serial dilutions of inactivated virus (10- 1 To 10- 9 ). At 37°C and 5% CO2 Incubate the culture plate for 96 hours to allow live virus to infect the cells. Virus replication was measured by observing the cytopathic effect (CPE) caused by the virus infecting MARC145 cells. For CPE, score each well containing virus dilution as positive or negative, and use Reed-Muench TCID50 Calculate and use the number of positive or negative wells of each dilution to determine TCID50 /mL. The simulated inactivated virus is treated with the same medium and temperature conditions as the inactivated virus (no inactivating agent is added). MARC145 cells cultured on T-225 flasks were used to perform three consecutive blind transmissions of simulated inactivated virus, inactivated virus, and only control medium. Add inactivated samples and control samples to the 2-day culture of MARC145 cells (add 3 mL of sample to 27 mL of medium) at 37°C and 5% CO2 Cultivate for 7 days. Visually check for the presence of CPE in the flask. For CPE, each flask is scored as positive (+) or negative (-) and recorded. Collect the supernatant from the flask and add it to a new set of T-225 flasks containing a 2-day culture of MARC-145 cells, and incubate at 37°C and 5% CO2 Cultivate for 7 days. Repeat the above procedure twice to obtain CPE observations for 3 consecutive viral generations. The samples stored at 2 different temperatures as shown were used to determine the PRRS virus potency at all collection time points during the 96-hour incubation period. 0 indicates that the power price is not detectable or not by the PRRS live virus TCID50 Determination analysis determination. The results presented in Table 5 show that the addition of 40 mM in the 2-step process can achieve a power value of at least 8 log10 TCID50 In the case of /mL, the PRRS virus is not activated. Higher potency indicates that the MBCD is not completely inactivated in the case of 20 mM MBCD, but after the second addition of MBCD to increase the concentration to 40 mM, the virus line is not detectable. These results also show that time is a less important contributor to inactivation. Given the MBCD concentration, longer incubation time did not increase the degree of inactivation. After 15 minutes of incubation, MBCD at a concentration of 20 mM reduced live viruses by approximately 2.5 log, and the level of detected live viruses remained unchanged for 24 hours. Adding 20 mM MBCD for the second time after 24 hours of incubation prevents the virus from being activated to an undetectable level at the next sample collection time point (30 hours). Differences in viability were also observed in the presence of 40 mM MBCD at different storage temperatures. The time-point samples with detectable live virus stored at -70°C have at least 100 times less virus than those at 4°C. table 5
Figure 105144117-A0304-0005
 a Log10 TCID50 /mLInstance 4 The goal of this study is to evaluate the safety and efficacy of experimental inactivated PRRSV vaccines in vaccination challenge studies. The vaccine is evaluated based on its ability to reduce lung disease and reduce the level of viremia. Vaccine characteristics of interest include storage temperature, dosage (1 vs. 2), and adjuvant addition. The study was conducted in the BSL-2 facility of Veterinary Resources, Inc. (Cambridge, Iowa). At Iowa State University Veterinary Diagnostic Laboratory (Ames, Iowa) and South Dakota State University Veterinary Diagnostic Laboratory (Brookings, South Dakota) Perform laboratory analysis. Sixty (60) clinically healthy weaned piglets about 3 weeks of age that were seronegative for PRRSV participated in the study. Sort the piglets by decreasing body weight and form 10 blocks with 6 animals in each block. The piglets were randomly assigned to one of the six treatment groups (10 piglets per group). The treatment group included an unvaccinated control group (T01) and five vaccinated experimentally inactivated PRRSV vaccine groups. The vaccine is not activated by means of delipidation using methyl-β-cyclodextrin (MBCD). The group T02 and group T03 vaccines do not contain adjuvants and are stored at 2°C to 8°C (T02) or -20°C (T03). Group T04 and Group T05 vaccines contain MONTANIDE™ Gel 01 adjuvant (Seppic), and administered once (T04) or twice (T05). Group T06 vaccine contains MONTANIDE™ IMS 1313 VGN PR adjuvant (Seppic) and was given twice. Standard commercial medicinal (CTC/DENAGARD)® ) Open feed (NRC, 2012) feed the piglets ad libitum. The piglets drank clean drinking water freely through the nipple drinker. On the 10th day after vaccination (DPV), use EXCEDE® Process all study piglets. In addition, use a single dose of VITAL E® -500 and EXCEDE® 21 DPV is another process to process all piglets. Administer all medicines according to label instructions.vaccine The PRRSV virus strain SD 11-21 (substitution degree 84) was used as the antigen in the current study. SD11-21 is a wild virus strain adapted to grow in the cell culture of MARC-145 cell line after 84 subcultures, three plaque purification and sucrose gradient centrifugation, as applied for on February 18, 2016 It is described in the same application US 62/296,658. In a 5 L BioBLU disposable bioreactor (Eppendorf, Hamburg, Germany) inoculated with MARC-145 connected to HILLEX II microcarriers (Pall Corporation, Port Washington, NY), it was supplemented with 2% fetal bovine serum (Sigma Louis, MO) and 50 ug/mL Gentamycin (Life Technologies, Grand Island, NY) in OPTI-MEM medium (Life Technologies, Grand Island, NY) to produce the 85th generation ( p85). As determined by the virus titration analysis described in Example 3, the power value of the collected p85 virus stock solution was 7.3 log10 TCID50 . In a 1 L bottle, add 19.7 mL of 300 mM MBCD (Sigma, St. Louis, MO) stock solution to 300 mL of SD11-21 virus stock solution. The mixture was incubated for 24 hours at room temperature with constant mixing at 50 rpm. After 24 hours of incubation, an additional 9.8 mL of MBCD stock solution was added, and the mixture was incubated for an additional 24 hours at room temperature with constant mixing at 50 rpm. In the case of a total of 48 hours of room temperature incubation, the final concentration of MBCD is 30 mM. The stock solution of simulated inactivated virus is also prepared, and the same amount of water is added instead of MBCD. The incubation time and mixing are the same as the inactivated virus. The simulated inactivated virus was used as a control group for virus measurement analysis. After treatment, simulate inactivated virus with 6.5 log10 TCID50 /mL exists, but no live virus is detected in the MBCD inactivated virus solution. The virus stock solution was stored at 4°C before further use. The vaccine is formulated to include stabilizers and preservatives. OPTI-MEM® Medium with reduced serum I was used as a blending diluent. Adjuvants include commercially available formulations of MONTANIDE™ Gel 01 (Seppic) and MONTANIDE™ 1313 VG N PR (Seppic). The control product was a commercially available preparation of phosphate buffered saline (PBS) (Corning Cellgro, Mediatech Inc.). The adjuvant-free vaccine contains delipidated virus, 25% stabilizer B, 25 μg/ml gentamicin as a preservative, and a blended diluent. The adjuvanted vaccine contains 20% of the designated adjuvant. Stabilizer B contains 2.5% D-mannitol (Sigma), 1.2% Type A gelatin (Fisher, Pittsburgh, PA), 1% NZ amine casein hydrolysate (Sigma), 5% sucrose (Sigma) and pH 7.0 to 7.2 It contains 6.2% trehalose (Fisher) ultrapure water (Life Technologies).Vaccinations and attacks Each of the 60 piglets participating in the study was vaccinated on the 35th day before challenge. The piglets in T01 to T05 were injected with a 1.0 mL intramuscular dose of the designated therapeutic agent on the right side of the neck. Inject 1.5 mL into the piglet in T06. The piglets in T01 to T03 and T05 and T06 were vaccinated again in the same way after the 14th day (the -21st day before challenge). At 0 DPC, the challenge material was prepared by thawing a frozen aliquot of the virus strain NADC-20 immediately before challenge. After being diluted in Minimum Essential Medium Eagle with Earle's salts and L-glutamic acid (MEM) from Mediatech, Inc., the power value was determined to be 1.26× 105 . 0 TCID50 /ml. Each piglet is physically constrained to face up in order to attack. A 3 mL non-luer (luer) locking syringe was used to deliver a 2 mL dose intranasally, approximately 1 mL per nostril.result At 14 DPC, the animal was humanely euthanized at each point. The lungs were excised and scored by research investigators who were unaware of the treatment. The overall characteristic lesions caused by PRRSV in each of the seven lung lobes were detected by visual inspection and palpation. The amount of lesion/consolidation in each lobe is scored as the actual value between 0 and 100% of the lobe. Enter the score of each lung lobe into the weight formula to calculate the percentage of lung lesions. Calculate the percentage of total lung lesions according to the following formula: Percentage of total lung lesions = {(0.10 × left top) + (0.10 × left heart) + (0.25 × left diaphragm) + (0.10 × right top) + (0.10 × right heart ) + (0.25 × right diaphragm) + (0.10 × middle)}. In addition, before further analysis, the square root of arcsine was used to convert the percentage of total lung lesions. The transformed data is analyzed by a mixed linear model including fixed processing factors (mixed procedures in SAS) and random block factors. The results of the statistical analysis of lung lesion scores are summarized in Table 6. The main treatment effect is systematically significant (P = 0.0003). The comparison with the control group (T01) showed that the Gel 01 adjuvant group (T04 and T05) had significantly lower (P <0.05) lung damage %. In addition, the non-adjuvant vaccine (T02) administered twice and stored at 2°C to 8°C produced significantly lower (T02) compared to the similar group (T03) treated with the vaccine stored at -20°C ( P <0.05) lesion score. Table 6. Average lung lesion score-lung damage after arcsine conversion% (main treatment effect: P = 0.0003)
Figure 105144117-A0304-0006
 *In the case of P <0.05, T01 is significantly different from T04 and T051 Unconverted average2 The average value after reverse conversion. Blood samples were collected on the -35th and -21st days before the challenge to determine the level of viremia. In addition, blood samples were collected at 0, 3, 7, 10, and 14 DPC to determine the level of viremia. Use qRT-PCR technology to test the sample for the presence of PRRS virus nucleic acid. In the case of non-normal distribution, convert serological and viremia data to log before analysis10 unit. Use repeated measurement mixed models (mixed procedures) to analyze the converted values. The statistical model includes processing, time, and the interaction of processing×time as a fixed factor. Blocks are included in the model as random factors. If the treatment × time interaction is significant (P <0.05), evaluate the effect within the treatment time. If the interaction is not significant, the main treatment effect is assessed. Shows the smallest mean square value (reversely converted) and standard error. The analysis of viremia levels is summarized in Table 7. No viremia was observed in any piglets prior to the PRRSV challenge, thus confirming that the vaccine virus line is not activated. The time point before the attack was excluded from the statistical analysis. After the attack, the treatment × days interaction was not significant (P = 0.0974), so the main treatment effect was evaluated. The treatment effect was significant (P = 0.0107). The comparison with the control group (T01) showed that all adjuvant vaccine groups (T04, T05 and T06) were significantly lower (P <0.05). None of the previous comparisons are statistically significant. Table 7. Viremia-Geometric mean of PRRSV genome copies/ml at 3, 7, 10, and 14 DPC (by log10 Conversion) (main treatment effect: P = 0.011; treatment × days interaction: P = 0.097)
Figure 105144117-A0304-0007
 *In the case of P<0.05, T01 is significantly different from T04, T05, and T06.1 Unconverted log10 average value2 Average value after reverse conversionin conclusion Compared with the control group, vaccinating piglets with Gel 01 adjuvant vaccine inactivated by MBCD at one or two doses effectively reduced both lung lesions and viremia levels. In piglets vaccinated with other MBCD-inactivated vaccines (T02 and T06) stored at 2°C to 8°C, lung lesions were numerically reduced. Due to changes in lung lesion scores, these differences are not significant at the P <0.05 level. Contains adjuvant (MONTANIDE™ All MBCD vaccines of Gel 01 or 1313) significantly reduce viremia. In future studies, when the NADC-20 virus strain is used as a challenge substance to detect significant biological differences between lung lesions and viremia, a larger sample size may be required. Storage temperature seems to have an impact on vaccine efficacy. Compared to vaccines stored at 2°C to 8°C, storing vaccines at -20°C has a negative impact on vaccine efficacy. In piglets vaccinated with the MBCD-inactivated vaccine containing Gel 01 adjuvant, adding additional vaccination 14 days after the first vaccination did not further improve the vaccine efficacy. Including Gel 01 adjuvant in vaccines that are not activated by MBCD does not affect vaccine efficacy. For example, after the second vaccination, Seppic MONTANIDE™ In the Gel 01 adjuvant group, there was no systemic reaction after vaccination, no injection site lesions, and only a brief (1 or 2 days) fever reaction. It was confirmed that the MBCD-inactivated vaccine is safe for growing pigs.

Claims (15)

一種製備不活化包膜病毒之方法，該方法包含：將包含包膜病毒之溶液與第一甲基β-環糊精(MBCD)溶液混合，獲得第一混合物，其中該MBCD之濃度為5mM至100mM；以第一時間段培育該第一混合物；將來自該第一混合物之該包膜病毒與第二MBCD溶液混合，獲得第二混合物；以及以第二時間段培育該第二混合物。 A method of preparing an inactivated enveloped virus, the method comprising: mixing a solution containing enveloped virus with a first methyl β-cyclodextrin (MBCD) solution to obtain a first mixture, wherein the concentration of the MBCD is 5 mM to 100 mM; incubate the first mixture for a first period of time; mix the enveloped virus from the first mixture with a second MBCD solution to obtain a second mixture; and incubate the second mixture for a second period of time. 如請求項1之方法，其中該第一混合物中MBCD之濃度為20mM至40mM。 The method of claim 1, wherein the concentration of MBCD in the first mixture is 20 mM to 40 mM. 如請求項1或2之方法，其中該第二混合物中MBCD之濃度為10mM至100mM。 The method of claim 1 or 2, wherein the concentration of MBCD in the second mixture is 10 mM to 100 mM. 如請求項1或2之方法，其中該第二混合物中MBCD之濃度為30mM至50mM。 The method of claim 1 or 2, wherein the concentration of MBCD in the second mixture is 30 mM to 50 mM. 如請求項1或2之方法，其中該第一時間段為15分鐘至24小時。 Such as the method of claim 1 or 2, wherein the first time period is 15 minutes to 24 hours. 如請求項1或2之方法，其中該第一時間段為4小時至24小時。 Such as the method of claim 1 or 2, wherein the first time period is 4 hours to 24 hours. 如請求項1或2之方法，其中該第二時間段為4小時至48小時。 Such as the method of claim 1 or 2, wherein the second time period is 4 hours to 48 hours. 如請求項1或2之方法，其中該第二時間段為24小時、36小時或48小時。 Such as the method of claim 1 or 2, wherein the second time period is 24 hours, 36 hours or 48 hours. 如請求項1或2之方法，其中該第一時間段及該第二時間段中之至少一者之溫度為20℃至25℃。 Such as the method of claim 1 or 2, wherein the temperature of at least one of the first time period and the second time period is 20°C to 25°C. 如請求項9之方法，其中該溫度為22℃至24℃。 Such as the method of claim 9, wherein the temperature is 22°C to 24°C. 如請求項1或2之方法，其中在獲得該第二混合物之步驟之前將該包膜病毒與該第一混合物分離。 The method of claim 1 or 2, wherein the enveloped virus is separated from the first mixture before the step of obtaining the second mixture. 如請求項1或2之方法，其中該包膜病毒為豬繁殖與呼吸症候群(PRRS)病毒。 The method of claim 1 or 2, wherein the enveloped virus is a porcine reproductive and respiratory syndrome (PRRS) virus. 如請求項1或2之方法，其中該第一時間段及該第二時間段中之至少一者進一步包含攪拌該第一混合物及/或該第二混合物。 The method of claim 1 or 2, wherein at least one of the first time period and the second time period further comprises stirring the first mixture and/or the second mixture. 一種製備疫苗之方法，其包含：將包含包膜病毒之溶液與第一MBCD溶液混合以獲得第一混合物；該第一MBCD溶液含有5mM至100mM之MBCD濃度；以15分鐘至24小時之第一時間段培育該第一混合物；將來自該第一混合物之該包膜病毒與第二MBCD溶液混合以獲得第 二混合物；該第二MBCD溶液含有10mM至100mM之MBCD濃度；以4小時至48小時之第二時間段培育該第二混合物；分離該包膜病毒；及將該分離之包膜病毒與以下之至少一者組合：佐劑、穩定劑、防腐劑及摻合稀釋劑；其中該第一時間段及該第二時間段中之至少一者之溫度為20℃至25℃。 A method for preparing a vaccine, which comprises: mixing a solution containing enveloped viruses with a first MBCD solution to obtain a first mixture; the first MBCD solution contains an MBCD concentration of 5 mM to 100 mM; and a first MBCD concentration of 15 minutes to 24 hours Cultivate the first mixture for a period of time; mix the enveloped virus from the first mixture with the second MBCD solution to obtain the first mixture Two mixtures; the second MBCD solution contains an MBCD concentration of 10 mM to 100 mM; incubate the second mixture for a second period of 4 hours to 48 hours; isolate the enveloped virus; and the isolated enveloped virus and the following At least one combination: adjuvant, stabilizer, preservative, and blended diluent; wherein the temperature of at least one of the first time period and the second time period is 20°C to 25°C. 如請求項14之方法，其中該包膜病毒為豬繁殖與呼吸症候群(PRRS)病毒。 The method of claim 14, wherein the enveloped virus is Porcine Reproductive and Respiratory Syndrome (PRRS) virus.
TW105144117A 2016-01-27 2016-12-30 Inactivation of pathogens by delipidation TWI747866B (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201662287488P 2016-01-27 2016-01-27
US62/287,488 2016-01-27

Publications (2)

Publication Number Publication Date
TW201733615A TW201733615A (en) 2017-10-01
TWI747866B true TWI747866B (en) 2021-12-01

Family

ID=57915178

Family Applications (1)

Application Number Title Priority Date Filing Date
TW105144117A TWI747866B (en) 2016-01-27 2016-12-30 Inactivation of pathogens by delipidation

Country Status (19)

Country Link
US (1) US10420831B2 (en)
EP (1) EP3408280B1 (en)
JP (1) JP6648286B2 (en)
KR (1) KR102202475B1 (en)
CN (1) CN108602860B (en)
AR (1) AR107262A1 (en)
AU (1) AU2017211040B2 (en)
BR (1) BR112018014542A2 (en)
CA (1) CA3008965C (en)
CL (1) CL2018001978A1 (en)
DK (1) DK3408280T3 (en)
EA (1) EA038712B1 (en)
ES (1) ES2839623T3 (en)
MX (1) MX2018008365A (en)
NZ (1) NZ743601A (en)
PL (1) PL3408280T3 (en)
TW (1) TWI747866B (en)
WO (1) WO2017132059A1 (en)
ZA (1) ZA201804025B (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002000266A2 (en) 2000-06-29 2002-01-03 Lipid Sciences, Inc. A method of treating and preventing infectious diseases

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997039761A1 (en) * 1996-04-19 1997-10-30 Alpha Therapeutic Corporation A process for viral inactivation of lyophilized blood proteins
ZA988446B (en) 1997-09-18 2000-03-22 Res Dev Foundation Production of vaccines using arthropod vectored viruses.
US7407662B2 (en) 2000-06-29 2008-08-05 Lipid Sciences, Inc. Modified viral particles with immunogenic properties and reduced lipid content
US20090017069A1 (en) 2000-06-29 2009-01-15 Lipid Sciences, Inc. SARS Vaccine Compositions and Methods of Making and Using Them
CU23002A1 (en) 2000-12-01 2004-11-18 Ct Ingenieria Genetica Biotech METHOD OF OBTAINING ANTIGENIC AGGREGATES AND THEIR USE IN FORMULATIONS
US8613934B2 (en) 2004-03-22 2013-12-24 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Cellular and viral inactivation
JP2006328039A (en) * 2005-04-26 2006-12-07 Nippo Kagaku Kk Bird flu virus deactivator
WO2009014774A1 (en) * 2007-04-06 2009-01-29 Inviragen, Inc. Methods and compositions for live attenuated viruses
GB0720503D0 (en) 2007-10-22 2007-11-28 Angeletti P Ist Richerche Bio New compound
EP2062594A1 (en) * 2007-11-21 2009-05-27 Wyeth Farma, S.A. Bluetongue virus vaccine and immunogenic compositions, methods of use and methods of producing same
EP2841097B1 (en) * 2012-04-24 2022-11-16 Ohio State Innovation Foundation Compositions and methods for treating and preventing porcine reproductive and respiratory syndrome

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002000266A2 (en) 2000-06-29 2002-01-03 Lipid Sciences, Inc. A method of treating and preventing infectious diseases

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
B E Cham et al., Delipidation of a hepadnavirus: Viral inactivation and vaccine development. Journal of Virological Methods. 2006 Oct;137(1):160-163. Epub 2006 Jun 30.
B E Cham et al., Delipidation of a hepadnavirus: Viral inactivation and vaccine development. Journal of Virological Methods. 2006 Oct;137(1):160-163. Epub 2006 Jun 30.; *
Hiroshi Fujita et al., Methyl-beta cyclodextrin alters the production and infectivity of Sendai virus. Archives of Virology volume 156, 995–1005(2011). *

Also Published As

Publication number Publication date
KR20180096775A (en) 2018-08-29
EA201891371A1 (en) 2019-01-31
EP3408280A1 (en) 2018-12-05
JP6648286B2 (en) 2020-02-14
EA038712B1 (en) 2021-10-08
CN108602860B (en) 2023-05-26
KR102202475B1 (en) 2021-01-12
JP2019506860A (en) 2019-03-14
CA3008965C (en) 2021-04-13
WO2017132059A1 (en) 2017-08-03
MX2018008365A (en) 2018-09-17
CL2018001978A1 (en) 2018-10-26
AR107262A1 (en) 2018-04-11
AU2017211040B2 (en) 2020-10-08
ZA201804025B (en) 2020-08-26
US20180360945A1 (en) 2018-12-20
EP3408280B1 (en) 2020-11-25
DK3408280T3 (en) 2021-01-04
TW201733615A (en) 2017-10-01
PL3408280T3 (en) 2021-05-31
ES2839623T3 (en) 2021-07-05
CA3008965A1 (en) 2017-08-03
US10420831B2 (en) 2019-09-24
CN108602860A (en) 2018-09-28
AU2017211040A1 (en) 2018-06-28
BR112018014542A2 (en) 2018-12-11
NZ743601A (en) 2020-03-27

Similar Documents

Publication Publication Date Title
WO2008073490A1 (en) Purification of influenza viral antigens
CN104232594B (en) Recombination classes fowl type H1N1 inactivated influenza virus vaccines strain (JS40/PR8) and its preparation method and application
US20140286979A1 (en) Canine influenza recombinant virus, preparation method therefor and application thereof
DK3010537T3 (en) PROCEDURE FOR PREVENTING AGGREGATION OF VIRUS COMPONENTS
JP5298377B2 (en) Medium supplements for virus production
Tabynov et al. Immunogenic and protective properties of the first Kazakhstan vaccine against pandemic influenza A (H1N1) pdm09 in ferrets
CN106929480B (en) Porcine reproductive and respiratory syndrome virus strain and application thereof
US20120107354A1 (en) Viral vaccine and process for preparing the same
TWI747866B (en) Inactivation of pathogens by delipidation
ES2562818T3 (en) Flu virus inactivation procedure
JP6560056B2 (en) H3 equine influenza A virus
RU2323741C2 (en) Anti- bird flue inactivated emulsion vaccine
RU2681439C1 (en) Influenza virus the virus-like particle and its obtaining method
US20240024390A1 (en) Use of a birnavirus alone or in combimation therapy for the treatment of a disease caused by a nidovirus
US20150174236A1 (en) Viral vaccine and process for preparing the same
Amara Old, Running and Future Vaccines: Where are we
CN118345053A (en) Method for preparing high-titer recombinant influenza virus by using pseudovirus model and application of method
RU2404804C2 (en) Inactivated vaccine against hepatitis a virus
RU2480238C1 (en) Newcastle disease, reovirus tenosynovitis and avian metapneumoviral infection associated inactivated emulsion vaccine
RU2509571C1 (en) Method for preparing cattle parainfluenza-3 vaccine
CN113151189A (en) H9N2 avian influenza virus, inactivated vaccine and preparation method thereof
CN114410594A (en) Infectious bronchitis virus adapted to cell replication and proliferation and application thereof
Bosworth et al. Evaluation of a Virus-like Replicon Particle Vaccine Expressing Proteins of Swine Influenza Virus in Pigs With and Without Maternally Derived Antibodies
BRPI0618094B1 (en) METHOD FOR PREPARING A CELL CULTURE PRODUCT

Legal Events

Date Code Title Description
MM4A Annulment or lapse of patent due to non-payment of fees